Fermentive production of four carbon alcohols

ABSTRACT

Methods for the fermentative production of four carbon alcohols is provided. Specifically, butanol, preferably isobutanol is produced by the fermentative growth of a recombinant bacterium expressing an isobutanol biosynthetic pathway.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.12/939,284, filed Nov. 4, 2010, which is a continuation of U.S.application Ser. No. 11/586,315, filed Oct. 25, 2006, now U.S. Pat. No.7,851,188, issued Dec. 14, 2010, which claims priority under 35 U.S.C.§119 from U.S. Provisional Application Ser. No. 60/730,290, filed Oct.26, 2005, each of which are incorporated by reference in their entirety.

REFERENCE TO A SEQUENCE LISTING SUBMITTED ELECTRONICALLY VIA EFS-WEB

The content of the electronically submitted sequence listing (Name:CL3243USCNT6_SequenceListing_CORRECTED_ascii.txt, Size: 380,433 bytes;and Date of Creation: Oct. 28, 2013) is herein incorporated by referencein its entirety.

FIELD OF THE INVENTION

The invention relates to the field of industrial microbiology and theproduction of alcohols. More specifically, isobutanol is produced viaindustrial fermentation of a recombinant microorganism.

BACKGROUND OF THE INVENTION

Butanol is an important industrial chemical, useful as a fuel additive,as a feedstock chemical in the plastics industry, and as a foodgradeextractant in the food and flavor industry. Each year 10 to 12 billionpounds of butanol are produced by petrochemical means and the need forthis commodity chemical will likely increase.

Methods for the chemical synthesis of isobutanol are known, such as oxosynthesis, catalytic hydrogenation of carbon monoxide (Ullmann'sEncyclopedia of Industrial Chemistry, 6^(th) edition, 2003,Wiley-VCHVerlag GmbH and Co., Weinheim, Germany, Vol. 5, pp. 716-719)and Guerbet condensation of methanol with n-propanol (Carlini et al., J.Mol. Catal. A: Chem. 220:215-220 (2004)). These processes use startingmaterials derived from petrochemicals and are generally expensive andare not environmentally friendly. The production of isobutanol fromplant-derived raw materials would minimize green house gas emissions andwould represent an advance in the art.

Isobutanol is produced biologically as a by-product of yeastfermentation. It is a component of “fusel oil” that forms as a result ofincomplete metabolism of amino acids by this group of fungi. Isobutanolis specifically produced from catabolism of L-valine. After the aminegroup of L-valine is harvested as a nitrogen source, the resultingα-keto acid is decarboxylated and reduced to isobutanol by enzymes ofthe so-called Ehrlich pathway (Dickinson et al., J. Biol. Chem.273(40):25752-25756 (1998)). Yields of fusel oil and/or its componentsachieved during beverage fermentation are typically low. For example,the concentration of isobutanol produced in beer fermentation isreported to be less than 16 parts per million (Garcia et al., ProcessBiochemistry 29:303-309 (1994)). Addition of exogenous L-valine to thefermentation increases the yield of isobutanol, as described byDickinson et al., supra, wherein it is reported that a yield ofisobutanol of 3 g/L is obtained by providing L-valine at a concentrationof 20 g/L in the fermentation. However, the use of valine as afeed-stock would be cost prohibitive for industrial scale isobutanolproduction. The biosynthesis of isobutanol directly from sugars would beeconomically viable and would represent an advance in the art. Therehave been no reports of a recombinant microorganism designed to produceisobutanol.

There is a need, therefore, for an environmentally responsible,cost-effective process for the production of isobutanol as a singleproduct. The present invention addresses this need by providing arecombinant microbial production host that expresses an isobutanolbiosynthetic pathway.

SUMMARY OF THE INVENTION

The invention provides a recombinant microorganism having an engineeredisobutanol biosynthetic pathway. The engineered microorganism may beused for the commercial production of isobutanol. Accordingly, in oneembodiment the invention provides a recombinant microbial host cellcomprising at least one DNA molecule encoding a polypeptide thatcatalyzes a substrate to product conversion selected from the groupconsisting of:

i) pyruvate to acetolactate (pathway step a)

ii) acetolactate to 2,3-dihydroxyisovalerate (pathway step b)

iii) 2,3-dihydroxyisovalerate to α-ketoisovalerate (pathway step c)

iv) α-ketoisovalerate to isobutyraldehyde, (pathway step d), and

v) isobutyraldehyde to isobutanol; (pathway step e)

wherein the at least one DNA molecule is heterologous to said microbialhost cell and wherein said microbial host cell produces isobutanol.

In another embodiment, the invention provides a recombinant microbialhost cell comprising at least one DNA molecule encoding a polypeptidethat catalyzes a substrate to product conversion selected from the groupconsisting of:

i) pyruvate to acetolactate, (pathway step a)

ii) acetolactate to 2,3-dihydroxyisovalerate, (pathway step b)

iii) 2,3-dihydroxyisovalerate to α-ketoisovalerate, (pathway step c)

iv) α-ketoisovalerate to isobutyryl-CoA, (pathway step f)

v) isobutyryl-CoA to isobutyraldehyde, (pathway step g), and

vi) isobutyraldehyde to isobutanol; (pathway step e)

wherein the at least one DNA molecule is heterologous to said microbialhost cell and wherein said microbial host cell produces isobutanol.

In another embodiment, the invention provides a recombinant microbialhost cell comprising at least one DNA molecule encoding a polypeptidethat catalyzes a substrate to product conversion selected from the groupconsisting of:

i) pyruvate to acetolactate, (pathway step a)

ii) acetolactate to 2,3-dihydroxyisovalerate, (pathway step b)

iii) 2,3-dihydroxyisovalerate to α-ketoisovalerate, (pathway step c)

iv) α-ketoisovalerate to valine, (pathway step h)

v) valine to isobutylamine, (pathway step i)

vi) isobutylamine to isobutyraldehyde, (pathway step j), and

vii) isobutyraldehyde to isobutanol: (pathway step e)

wherein the at least one DNA molecule is heterologous to said microbialhost cell and wherein said microbial host cell produces isobutanol.

In another embodiment, the invention provides a method for theproduction of isobutanol comprising:

-   -   1) providing a recombinant microbial host cell comprising at        least one DNA molecule encoding a polypeptide that catalyzes a        substrate to product conversion selected from the group        consisting of:

i) pyruvate to acetolactate (pathway step a)

ii) acetolactate to 2,3-dihydroxyisovalerate (pathway step b)

iii) 2,3-dihydroxyisovalerate to α-ketoisovalerate (pathway step c)

iv) α-ketoisovalerate to isobutyraldehyde, (pathway step d), and

v) isobutyraldehyde to isobutanol; (pathway step e)

wherein the at least one DNA molecule is heterologous to said microbialhost cell; and

-   -   2) contacting the host cell of (i) with a fermentable carbon        substrate in a fermentation medium under conditions whereby        isobutanol is produced.

In another embodiment, the invention provides a method for theproduction of isobutanol comprising:

-   -   1) providing a recombinant microbial host cell comprising at        least one DNA molecule encoding a polypeptide that catalyzes a        substrate to product conversion selected from the group        consisting of:

i) pyruvate to acetolactate, (pathway step a)

ii) acetolactate to 2,3-dihydroxyisovalerate, (pathway step b)

iii) 2,3-dihydroxyisovalerate to α-ketoisovalerate, (pathway step c)

iv) α-ketoisovalerate to isobutyryl-CoA, (pathway step f)

v) isobutyryl-CoA to isobutyraldehyde, (pathway step g), and

vi) isobutyraldehyde to isobutanol; (pathway step e)

wherein the at least one DNA molecule is heterologous to said microbialhost cell; and

-   -   2) contacting the host cell of (i) with a fermentable carbon        substrate in a fermentation medium under conditions whereby        isobutanol is produced.

In another embodiment, the invention provides a method for theproduction of isobutanol comprising:

-   -   1) providing a recombinant microbial host cell comprising at        least one DNA molecule encoding a polypeptide that catalyzes a        substrate to product conversion selected from the group        consisting of:

i) pyruvate to acetolactate, (pathway step a)

ii) acetolactate to 2,3-dihydroxyisovalerate, (pathway step b)

iii) 2,3-dihydroxyisovalerate to α-ketoisovalerate, (pathway step c)

iv) α-ketoisovalerate to valine, (pathway step h)

v) valine to isobutylamine, (pathway step i)

vi) isobutylamine to isobutyraldehyde, (pathway step j), and

vii) isobutyraldehyde to isobutanol: (pathway step e)

wherein the at least one DNA molecule is heterologous to said microbialhost cell; and

-   -   2) contacting the host cell of (i) with a fermentable carbon        substrate in a fermentation medium under conditions whereby        isobutanol is produced.

In an alternate embodiment the invention provides an isobutanolconstaining fermentation medium produced by the methods of theinvention.

BRIEF DESCRIPTION OF THE FIGURES AND SEQUENCE DESCRIPTIONS

The invention can be more fully understood from the following detaileddescription, FIGURE, and the accompanying sequence descriptions, whichform a part of this application.

FIG. 1 shows four different isobutanol biosynthetic pathways. The stepslabeled “a”, “b”, “c”, “d”, “e”, “f”, “g”, “h”, “i”, “j” and “k”represent the substrate to product conversions described below.

The following sequences conform with 37 C.F.R. §1.821-1.825(“Requirements for Patent Applications Containing Nucleotide Sequencesand/or Amino Acid Sequence Disclosures—the Sequence Rules”) and areconsistent with World Intellectual Property Organization (WIPO) StandardST.25 (2009) and the sequence listing requirements of the EPO and PCT(Rules 5.2 and 49.5(a-bis), and Section 208 and Annex C of theAdministrative Instructions). The symbols and format used for nucleotideand amino acid sequence data comply with the rules set forth in 37C.F.R. §1.822.

TABLE 1 Summary of Gene and Protein SEQ ID Numbers SEQ ID NO: SEQ IDNucleic NO: Description acid Peptide Klebsiella pneumoniae budB 1 2(acetolactate synthase) Bacillus subtilis alsS 78 178 (acetolactatesynthase) Lactococcus lactis als 179 180 (acetolactate synthase) E. coliilvC (acetohydroxy acid 3 4 reductoisomerase) S. cerevisiae ILV5 80 181(acetohydroxy acid reductoisomerase) M. maripaludis ilvC 182 183(Ketol-acid reductoisomerase) B. subtilis ilvC 184 185 (acetohydroxyacid reductoisomerase) E. coli ilvD (acetohydroxy acid 5 6 dehydratase)S. cerevisiae ILV3 83 186 (Dihydroxyacid dehydratase) M. maripaludisilvD 187 188 (Dihydroxy-acid dehydratase) B. subtilis ilvD 189 190(dihydroxy-acid dehydratase) Lactococcus lactis kivD (branched- 7 8chain α-keto acid decarboxylase), codon optimized Lactococcus lactiskivD (branched- 191 8 chain α-keto acid decarboxylase), Lactococcuslactis kdcA 192 193 (branched-chain alpha-ketoacid decarboxylase)Salmonella typhimurium 194 195 (indolepyruvate decarboxylase)Clostridium acetobutylicum pdc 196 197 (Pyruvate decarboxylase) E. coliyqhD (branched-chain alcohol 9 10 dehydrogenase) S. cerevisiae YPR1 198199 (2-methylbutyraldehyde reductase) S. cerevisiae ADH6 200 201(NADPH-dependent cinnamyl alcohol dehydrogenase) Clostridiumacetobutylicum bdhA 202 203 (NADH-dependent butanol dehydrogenase A)Clostridium acetobutylicum bdhB 158 204 Butanol dehydrogenase B.subtilis bkdAA 205 206 (branched-chain keto acid dehydrogenase E1subunit) B. subtilis bkdAB 207 208 (branched-chain alpha-keto aciddehydrogenase E1 subunit) B. subtilis bkdB 209 210 (branched-chainalpha-keto acid dehydrogenase E2 subunit) B. subtilis lpdV 211 212(branched-chain alpha-keto acid dehydrogenase E3 subunit) P. putidabkdA1 213 214 (keto acid dehydrogenase E1-alpha subunit) P. putida bkdA2215 216 (keto acid dehydrogenase E1-beta subunit) P. putida bkdB 217 218(transacylase E2) P. putida 1pdV 219 220 (lipoamide dehydrogenase) C.beijerinckii ald 221 222 (coenzyme A acylating aldehyde dehydrogenase)C. acetobutylicum adhe1 223 224 (aldehyde dehydrogenase) C.acetobutylicum adhe 225 226 (alcohol-aldehyde dehydrogenase) P. putidanahO 227 228 (acetaldehyde dehydrogenase) T. thermophilus 229 230(acetaldehyde dehydrogenase) E. coli avtA 231 232 (valine-pyruvatetransaminase) B. licheniformis avtA 233 234 (valine-pyruvatetransaminase) E. coli ilvE 235 236 (branched chain amino acidaminotransferase) S. cerevisiae BAT2 237 238 (branched chain amino acidaminotransferase) M. thermoautotrophicum 239 240 (branched chain aminoacid aminotransferase) S. coelicolor 241 242 (valine dehydrogenase) B..subtilis bcd 243 244 (leucine dehydrogenase) S. viridifaciens 245 246(valine decarboxyase) A. denitrificans aptA 247 248 (omega-aminoacid:pyruvate transaminase) R. eutropha 249 250 (alanine-pyruvatetransaminase) S. oneidensis 251 252 (beta alanine-pyruvate transaminase)P. putida 253 254 (beta alanine-pyruvate transaminase) S. cinnamonensisicm 255 256 (isobutyrl-CoA mutase) S. cinnamonensis icmB 257 258(isobutyrl-CoA mutase) S. coelicolor SCO5415 259 260 (isobutyrl-CoAmutase) S. coelicolor SCO4800 261 262 (isobutyrl-CoA mutase) S.avermitilis icmA 263 264 (isobutyrl-CoA mutase) S. avermitilis icmB 265266 (isobutyrl-CoA mutase)

SEQ ID NOs:11-38, 40-69, 72-75, 85-138, 144, 145, 147-157, 159-176 arethe nucleotide sequences of oligonucleotide cloning, screening orsequencing primers used in the Examples described herein.

SEQ ID NO:39 is the nucleotide sequence of the cscBKA gene clusterdescribed in Example 16.

SEQ ID NO:70 is the nucleotide sequence of the glucose isomerasepromoter 1.6GI described in Example 13.

SEQ ID NO:71 is the nucleotide sequence of the 1.5GI promoter describedin Example 13.

SEQ ID NO:76 is the nucleotide sequence of the GPD promoter described inExample 17.

SEQ ID NO:77 is the nucleotide sequence of the CYC1 terminator describedin Example 17.

SEQ ID NO:79 is the nucleotide sequence of the FBA promoter described inExample 17.

SEQ ID NO:81 is the nucleotide sequence of ADH1 promoter described inExample 17.

SEQ ID NO:82 is the nucleotide sequence of ADH1 terminator described inExample 17.

SEQ ID NO:84 is the nucleotide sequence of GPM promoter described inExample 17.

SEQ ID NO:139 is the amino acid sequence of sucrose hydrolase (CscA).

SEQ ID NO:140 is the amino acid sequence of D-fructokinase (CscK).

SEQ ID NO:141 is the amino acid sequence of sucrose permease (CscB).

SEQ ID NO:142 is the nucleotide sequence of plasmid pFP988DssPspacdescribed in Example 20.

SEQ ID NO:143 is the nucleotide sequence of plasmid pFP988DssPgroEdescribed in Example 20.

SEQ ID NO:146 is the nucleotide sequence of the pFP988Dss vectorfragment described in Example 20.

SEQ ID NO:177 is the nucleotide sequence of the pFP988 integrationvector described in Example 21.

SEQ ID NO:267 is the nucleotide sequence of plasmid pC194 described inExample 21.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods for the production ofisobutanol using recombinant microorganisms. The present invention meetsa number of commercial and industrial needs. Butanol is an importantindustrial commodity chemical with a variety of applications, where itspotential as a fuel or fuel additive is particularly significant.Although only a four-carbon alcohol, butanol has an energy contentsimilar to that of gasoline and can be blended with any fossil fuel.Butanol is favored as a fuel or fuel additive as it yields only CO₂ andlittle or no SO_(X) or NO_(X) when burned in the standard internalcombustion engine. Additionally butanol is less corrosive than ethanol,the most preferred fuel additive to date.

In addition to its utility as a biofuel or fuel additive, butanol hasthe potential of impacting hydrogen distribution problems in theemerging fuel cell industry. Fuel cells today are plagued by safetyconcerns associated with hydrogen transport and distribution. Butanolcan be easily reformed for its hydrogen content and can be distributedthrough existing gas stations in the purity required for either fuelcells or vehicles.

Finally the present invention produces isobutanol from plant derivedcarbon sources, avoiding the negative environmental impact associatedwith standard petrochemical processes for butanol production.

The following definitions and abbreviations are to be used for theinterpretation of the claims and the specification.

The term “invention” or “present invention” as used herein is anon-limiting term and is not intended to refer to any single embodimentof the particular invention but encompasses all possible embodiments asdescribed in the specification and the claims.

The term “isobutanol biosynthetic pathway” refers to an enzyme pathwaysto produce isobutanol.

The terms “acetolactate synthase” and “acetolactate synthetase” are usedintechangeably herein to refer to an enzyme that catalyzes theconversion of pyruvate to acetolactate and CO₂. Preferred acetolactatesynthases are known by the EC number 2.2.1.6 (Enzyme Nomenclature 1992,Academic Press, San Diego). These enzymes are available from a number ofsources, including, but not limited to, Bacillus subtilis (GenBank Nos:CAB15618 (SEQ ID NO:178), Z99122 (SEQ ID NO:78), NCBI (National Centerfor Biotechnology Information) amino acid sequence, NCBI nucleotidesequence, respectively), Klebsiella pneumoniae (GenBank Nos: AAA25079(SEQ ID NO:2), M73842 (SEQ ID NO:1)), and Lactococcus lactis (GenBankNos: AAA25161 (SEQ ID NO:180), L16975 (SEQ ID NO:179)).

The terms “acetohydroxy acid isomeroreductase” and “acetohydroxy acidreductoisomerase” are used interchangeably herein to refer to an enzymethat catalyzes the conversion of acetolactate to2,3-dihydroxyisovalerate using NADPH (reduced nicotinamide adeninedinucleotide phosphate) as an electron donor. Preferred acetohydroxyacid isomeroreductases are known by the EC number 1.1.1.86 and sequencesare available from a vast array of microorganisms, including, but notlimited to, Escherichia coli (GenBank Nos: NP_(—)418222 (SEQ ID NO:4),NC_(—)000913 (SEQ ID NO:3)), Saccharomyces cerevisiae (GenBank Nos:NP_(—)013459 (SEQ ID NO:181), NC_(—)001144 (SEQ ID NO:80)),Methanococcus maripaludis (GenBank Nos: CAF30210 (SEQ ID NO:183),BX957220 (SEQ ID NO:182)), and Bacillus. subtilis (GenBank Nos: CAB14789(SEQ ID NO:185), Z99118 (SEQ ID NO:184)).

The term “acetohydroxy acid dehydratase” refers to an enzyme thatcatalyzes the conversion of 2,3-dihydroxyisovalerate toα-ketoisovalerate. Preferred acetohydroxy acid dehydratases are known bythe EC number 4.2.1.9. These enzymes are available from a vast array ofmicroorganisms, including, but not limited to, E. coli (GenBank Nos:YP_(—)026248 (SEQ ID NO:6), NC_(—)000913 (SEQ ID NO:5)), S. cerevisiae(GenBank Nos: NP_(—)012550 (SEQ ID NO:186), NC_(—)001142 (SEQ IDNO:83)), M. maripaludis (GenBank Nos: CAF29874 (SEQ ID NO:188), BX957219(SEQ ID NO:187)), and B. subtilis (GenBank Nos: CAB14105 (SEQ IDNO:190), Z99115 (SEQ ID NO:189)).

The term “branched-chain α-keto acid decarboxylase” refers to an enzymethat catalyzes the conversion of α-ketoisovalerate to isobutyraldehydeand CO₂. Preferred branched-chain α-keto acid decarboxylases are knownby the EC number 4.1.1.72 and are available from a number of sources,including, but not limited to, Lactococcus lactis (GenBank Nos: AAS49166(SEQ ID NO:193), AY548760 (SEQ ID NO:192); CAG34226 (SEQ ID NO:8),AJ746364 (SEQ ID NO:191), Salmonella typhimurium (GenBank Nos:NP_(—)461346 (SEQ ID NO:195), NC_(—)003197 (SEQ ID NO:194)), andClostridium acetobutylicum (GenBank Nos: NP_(—)149189 (SEQ ID NO:197),NC_(—)001988 (SEQ ID NO:196)).

The term “branched-chain alcohol dehydrogenase” refers to an enzyme thatcatalyzes the conversion of isobutyraldehyde to isobutanol. Preferredbranched-chain alcohol dehydrogenases are known by the EC number1.1.1.265, but may also be classified under other alcohol dehydrogenases(specifically, EC 1.1.1.1 or 1.1.1.2). These enzymes utilize NADH(reduced nicotinamide adenine dinucleotide) and/or NADPH as electrondonor and are available from a number of sources, including, but notlimited to, S. cerevisiae (GenBank Nos: NP_(—)010656 (SEQ ID NO:199),NC_(—)001136 (SEQ ID NO:198); NP_(—)014051 (SEQ ID NO:201) NC_(—)001145(SEQ ID NO:200)), E. coli (GenBank Nos: NP_(—)417484 (SEQ ID NO:10),NC_(—)000913 (SEQ ID NO:9)), and C. acetobutylicum (GenBank Nos:NP_(—)349892 (SEQ ID NO:203), NC_(—)003030 (SEQ ID NO:202); NP_(—)349891(SEQ ID NO:204), NC_(—)003030 (SEQ ID NO:158)).

The term “branched-chain keto acid dehydrogenase” refers to an enzymethat catalyzes the conversion of α-ketoisovalerate to isobutyryl-CoA(isobutyryl-coenzyme A), using NAD⁺ (nicotinamide adenine dinucleotide)as electron acceptor. Preferred branched-chain keto acid dehydrogenasesare known by the EC number 1.2.4.4. These branched-chain keto aciddehydrogenases are comprised of four subunits and sequences from allsubunits are available from a vast array of microorganisms, including,but not limited to, B. subtilis (GenBank Nos: CAB14336 (SEQ ID NO:206),Z99116 (SEQ ID NO:205); CAB14335 (SEQ ID NO:208), Z99116 (SEQ IDNO:207); CAB14334 (SEQ ID NO:210), Z99116 (SEQ ID NO:209); and CAB14337(SEQ ID NO:212), Z99116 (SEQ ID NO:211)) and Pseudomonas putida (GenBankNos: AAA65614 (SEQ ID NO:214), M57613 (SEQ ID NO:213); AAA65615 (SEQ IDNO:216), M57613 (SEQ ID NO:215); AAA65617 (SEQ ID NO:218), M57613 (SEQID NO:217); and AAA65618 (SEQ ID NO:220), M57613 (SEQ ID NO:219)).

The term “acylating aldehyde dehydrogenase” refers to an enzyme thatcatalyzes the conversion of isobutyryl-CoA to isobutyraldehyde, usingeither NADH or NADPH as electron donor. Preferred acylating aldehydedehydrogenases are known by the EC numbers 1.2.1.10 and 1.2.1.57. Theseenzymes are available from multiple sources, including, but not limitedto, Clostridium beijerinckii (GenBank Nos: AAD31841 (SEQ ID NO:222),AF157306 (SEQ ID NO:221)), C. acetobutylicum (GenBank Nos: NP_(—)149325(SEQ ID NO:224), NC_(—)001988 (SEQ ID NO:223); NP_(—)149199 (SEQ IDNO:226), NC_(—)001988 (SEQ ID NO:225)), P. putida (GenBank Nos: AAA89106(SEQ ID NO:228), U13232 (SEQ ID NO:227)), and Thermus thermophilus(GenBank Nos: YP_(—)145486 (SEQ ID NO:230), NC_(—)006461 (SEQ IDNO:229)).

The term “transaminase” refers to an enzyme that catalyzes theconversion of α-ketoisovalerate to L-valine, using either alanine orglutamate as amine donor. Preferred transaminases are known by the ECnumbers 2.6.1.42 and 2.6.1.66. These enzymes are available from a numberof sources. Examples of sources for alanine-dependent enzymes include,but are not limited to, E. coli (GenBank Nos: YP_(—)026231 (SEQ IDNO:232), NC_(—)000913 (SEQ ID NO:231)) and Bacillus licheniformis(GenBank Nos: YP_(—)093743 (SEQ ID NO:234), NC_(—)006322 (SEQ IDNO:233)). Examples of sources for glutamate-dependent enzymes include,but are not limited to, E. coli (GenBank Nos: YP_(—)026247 (SEQ IDNO:236), NC_(—)000913 (SEQ ID NO:235)), S. cerevisiae (GenBank Nos:NP_(—)012682 (SEQ ID NO:238), NC_(—)001142 (SEQ ID NO:237)) andMethanobacterium thermoautotrophicum (GenBank Nos: NP_(—)276546 (SEQ IDNO:240), NC_(—)000916 (SEQ ID NO:239)).

The term “valine dehydrogenase” refers to an enzyme that catalyzes theconversion of α-ketoisovalerate to L-valine, using NAD(P)H as electrondonor and ammonia as amine donor. Preferred valine dehydrogenases areknown by the EC numbers 1.4.1.8 and 1.4.1.9 and are available from anumber of sources, including, but not limited to, Streptomycescoelicolor (GenBank Nos: NP_(—)628270 (SEQ ID NO:242), NC_(—)003888 (SEQID NO:241)) and B. subtilis (GenBank Nos: CAB14339 (SEQ ID NO:244),Z99116 (SEQ ID NO:243)).

The term “valine decarboxylase” refers to an enzyme that catalyzes theconversion of L-valine to isobutylamine and CO₂. Preferred valinedecarboxylases are known by the EC number 4.1.1.14. These enzymes arefound in Streptomycetes, such as for example, Streptomyces viridifaciens(GenBank Nos: AAN10242 (SEQ ID NO:246), AY116644 (SEQ ID NO:245)).

The term “omega transaminase” refers to an enzyme that catalyzes theconversion of isobutylamine to isobutyraldehyde using a suitable aminoacid as amine donor. Preferred omega transaminases are known by the ECnumber 2.6.1.18 and are available from a number of sources, including,but not limited to, Alcaligenes denitrificans (AAP92672 (SEQ ID NO:248),AY330220 (SEQ ID NO:247)), Ralstonia eutropha (GenBank Nos: YP_(—)294474(SEQ ID NO:250), NC_(—)007347 (SEQ ID NO:249)), Shewanella oneidensis(GenBank Nos: NP_(—)719046 (SEQ ID NO:252), NC_(—)004347 (SEQ IDNO:251)), and P. putida (GenBank Nos: AAN66223 (SEQ ID NO:254), AE016776(SEQ ID NO:253)).

The term “isobutyryl-CoA mutase” refers to an enzyme that catalyzes theconversion of butyryl-CoA to isobutyryl-CoA. This enzyme uses coenzymeB₁₂ as cofactor. Preferred isobutyryl-CoA mutases are known by the ECnumber 5.4.99.13. These enzymes are found in a number of Streptomycetes,including, but not limited to, Streptomyces cinnamonensis (GenBank Nos:AAC08713 (SEQ ID NO:256), U67612 (SEQ ID NO:255); CAB59633 (SEQ IDNO:258), AJ246005 (SEQ ID NO:257)), S. coelicolor (GenBank Nos: CAB70645(SEQ ID NO:260), AL939123 (SEQ ID NO:259); CAB92663 (SEQ ID NO:262),AL939121 (SEQ ID NO:261)), and Streptomyces avermitilis (GenBank Nos:NP_(—)824008 (SEQ ID NO:264), NC_(—)003155 (SEQ ID NO:263); NP_(—)824637(SEQ ID NO:266), NC_(—)003155 (SEQ ID NO:265)).

The term “a facultative anaerobe” refers to a microorganism that cangrow in both aerobic and anaerobic environments.

The term “carbon substrate” or “fermentable carbon substrate” refers toa carbon source capable of being metabolized by host organisms of thepresent invention and particularly carbon sources selected from thegroup consisting of monosaccharides, oligosaccharides, polysaccharides,and one-carbon substrates or mixtures thereof.

The term “gene” refers to a nucleic acid fragment that is capable ofbeing expressed as a specific protein, optionally including regulatorysequences preceding (5′ non-coding sequences) and following (3′non-coding sequences) the coding sequence. “Native gene” refers to agene as found in nature with its own regulatory sequences. “Chimericgene” refers to any gene that is not a native gene, comprisingregulatory and coding sequences that are not found together in nature.Accordingly, a chimeric gene may comprise regulatory sequences andcoding sequences that are derived from different sources, or regulatorysequences and coding sequences derived from the same source, butarranged in a manner different than that found in nature. “Endogenousgene” refers to a native gene in its natural location in the genome ofan organism. A “foreign gene” or “heterologous gene” refers to a genenot normally found in the host organism, but that is introduced into thehost organism by gene transfer. Foreign genes can comprise native genesinserted into a non-native organism, or chimeric genes. A “transgene” isa gene that has been introduced into the genome by a transformationprocedure.

As used herein the term “coding sequence” refers to a DNA sequence thatcodes for a specific amino acid sequence. “Suitable regulatorysequences” refer to nucleotide sequences located upstream (5′ non-codingsequences), within, or downstream (3′ non-coding sequences) of a codingsequence, and which influence the transcription, RNA processing orstability, or translation of the associated coding sequence. Regulatorysequences may include promoters, translation leader sequences, introns,polyadenylation recognition sequences, RNA processing site, effectorbinding site and stem-loop structure.

The term “promoter” refers to a DNA sequence capable of controlling theexpression of a coding sequence or functional RNA. In general, a codingsequence is located 3′ to a promoter sequence. Promoters may be derivedin their entirety from a native gene, or be composed of differentelements derived from different promoters found in nature, or evencomprise synthetic DNA segments. It is understood by those skilled inthe art that different promoters may direct the expression of a gene indifferent tissues or cell types, or at different stages of development,or in response to different environmental or physiological conditions.Promoters which cause a gene to be expressed in most cell types at mosttimes are commonly referred to as “constitutive promoters”. It isfurther recognized that since in most cases the exact boundaries ofregulatory sequences have not been completely defined, DNA fragments ofdifferent lengths may have identical promoter activity.

The term “operably linked” refers to the association of nucleic acidsequences on a single nucleic acid fragment so that the function of oneis affected by the other. For example, a promoter is operably linkedwith a coding sequence when it is capable of effecting the expression ofthat coding sequence (i.e., that the coding sequence is under thetranscriptional control of the promoter). Coding sequences can beoperably linked to regulatory sequences in sense or antisenseorientation.

The term “expression”, as used herein, refers to the transcription andstable accumulation of sense (mRNA) or antisense RNA derived from thenucleic acid fragment of the invention. Expression may also refer totranslation of mRNA into a polypeptide.

As used herein the term “transformation” refers to the transfer of anucleic acid fragment into a host organism, resulting in geneticallystable inheritance. Host organisms containing the transformed nucleicacid fragments are referred to as “transgenic” or “recombinant” or“transformed” organisms.

The terms “plasmid”, “vector” and “cassette” refer to an extrachromosomal element often carrying genes which are not part of thecentral metabolism of the cell, and usually in the form of circulardouble-stranded DNA fragments. Such elements may be autonomouslyreplicating sequences, genome integrating sequences, phage or nucleotidesequences, linear or circular, of a single- or double-stranded DNA orRNA, derived from any source, in which a number of nucleotide sequenceshave been joined or recombined into a unique construction which iscapable of introducing a promoter fragment and DNA sequence for aselected gene product along with appropriate 3′ untranslated sequenceinto a cell. “Transformation cassette” refers to a specific vectorcontaining a foreign gene and having elements in addition to the foreigngene that facilitates transformation of a particular host cell.“Expression cassette” refers to a specific vector containing a foreigngene and having elements in addition to the foreign gene that allow forenhanced expression of that gene in a foreign host.

As used herein the term “codon degeneracy” refers to the nature in thegenetic code permitting variation of the nucleotide sequence withouteffecting the amino acid sequence of an encoded polypeptide. The skilledartisan is well aware of the “codon-bias” exhibited by a specific hostcell in usage of nucleotide codons to specify a given amino acid.Therefore, when synthesizing a gene for improved expression in a hostcell, it is desirable to design the gene such that its frequency ofcodon usage approaches the frequency of preferred codon usage of thehost cell.

The term “codon-optimized” as it refers to genes or coding regions ofnucleic acid molecules for transformation of various hosts, refers tothe alteration of codons in the gene or coding regions of the nucleicacid molecules to reflect the typical codon usage of the host organismwithout altering the polypeptide encoded by the DNA.

Standard recombinant DNA and molecular cloning techniques used hereinare well known in the art and are described by Sambrook, J., Fritsch, E.F. and Maniatis, T., Molecular Cloning: A Laboratory Manual, SecondEdition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(1989) (hereinafter “Maniatis”); and by Silhavy, T. J., Bennan, M. L.and Enquist, L. W., Experiments with Gene Fusions, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1984); and by Ausubel, F. M.et al., Current Protocols in Molecular Biology, published by GreenePublishing Assoc. and Wiley-Interscience (1987).

Isobutanol Biosynthetic Pathways

Carbohydrate utilizing microorganisms employ the Embden-Meyerhof-Parnas(EMP) pathway, the Entner-Doudoroff pathway and the pentose phosphatecycle as the central, metabolic routes to provide energy and cellularprecursors for growth and maintenance. These pathways have in common theintermediate glyceraldehyde-3-phosphate and, ultimately, pyruvate isformed directly or in combination with the EMP pathway. Subsequently,pyruvate is transformed to acetyl-coenzyme A (acetyl-CoA) via a varietyof means. Acetyl-CoA serves as a key intermediate, for example, ingenerating fatty acids, amino acids and secondary metabolites. Thecombined reactions of sugar conversion to pyruvate produce energy (e.g.adenosine-5′-triphosphate, ATP) and reducing equivalents (e.g. reducednicotinamide adenine dinucleotide, NADH, and reduced nicotinamideadenine dinucleotide phosphate, NADPH). NADH and NADPH must be recycledto their oxidized forms (NAD⁺ and NADP⁺, respectively). In the presenceof inorganic electron acceptors (e.g. O₂, NO₃ ⁻ and SO₄ ²⁻), thereducing equivalents may be used to augment the energy pool;alternatively, a reduced carbon by-product may be formed.

The invention enables the production of isobutanol from carbohydratesources with recombinant microorganisms by providing four completereaction pathways, as shown in FIG. 1. Three of the pathways compriseconversion of pyruvate to isobutanol via a series of enzymatic steps.The preferred isobutanol pathway (FIG. 1, steps a to e), comprises thefollowing substrate to product conversions:

-   -   a) pyruvate to acetolactate, as catalyzed for example by        acetolactate synthase,    -   b) acetolactate to 2,3-dihydroxyisovalerate, as catalyzed for        example by acetohydroxy acid isomeroreductase,    -   c) 2,3-dihydroxyisovalerate to α-ketoisovalerate, as catalyzed        for example by acetohydroxy acid dehydratase,    -   d) α-ketoisovalerate to isobutyraldehyde, as catalyzed for        example by a branched-chain keto acid decarboxylase, and    -   e) isobutyraldehyde to isobutanol, as catalyzed for example by,        a branched-chain alcohol dehydrogenase.

This pathway combines enzymes known to be involved in well-characterizedpathways for valine biosynthesis (pyruvate to α-ketoisovalerate) andvaline catabolism (α-ketoisovalerate to isobutanol). Since many valinebiosynthetic enzymes also catalyze analogous reactions in the isoleucinebiosynthetic pathway, substrate specificity is a major consideration inselecting the gene sources. For this reason, the primary genes ofinterest for the acetolactate synthase enzyme are those from Bacillus(alsS) and Klebsiella (budB). These particular acetolactate synthasesare known to participate in butanediol fermentation in these organismsand show increased affinity for pyruvate over ketobutyrate (Gollop etal., J. Bacteriol. 172(6):3444-3449 (1990); Holtzclaw et al., J.Bacteriol. 121(3):917-922 (1975)). The second and third pathway stepsare catalyzed by acetohydroxy acid reductoisomerase and dehydratase,respectively. These enzymes have been characterized from a number ofsources, such as for example, E. coli (Chunduru et al., Biochemistry28(2):486-493 (1989); Flint et al., J. Biol. Chem. 268(29):14732-14742(1993)). The final two steps of the preferred isobutanol pathway areknown to occur in yeast, which can use valine as a nitrogen source and,in the process, secrete isobutanol. α-Ketoisovalerate can be convertedto isobutyraldehyde by a number of keto acid decarboxylase enzymes, suchas for example pyruvate decarboxylase. To prevent misdirection ofpyruvate away from isobutanol production, a decarboxylase with decreasedaffinity for pyruvate is desired. So far, there are two such enzymesknown in the art (Smit et al., Appl. Environ. Microbiol. 71(1):303-311(2005); de la Plaza et al., FEMS Microbiol. Lett. 238(2):367-374(2004)). Both enzymes are from strains of Lactococcus lactis and have a50-200-fold preference for ketoisovalerate over pyruvate. Finally, anumber of aldehyde reductases have been identified in yeast, many withoverlapping substrate specificity. Those known to prefer branched-chainsubstrates over acetaldehyde include, but are not limited to, alcoholdehydrogenase VI (ADH6) and Ypr1 p (Larroy et al., Biochem. J. 361(Pt1):163-172 (2002); Ford et al., Yeast 19(12):1087-1096 (2002)), both ofwhich use NADPH as electron donor. An NADPH-dependent reductase, YqhD,active with branched-chain substrates has also been recently identifiedin E. coli (Sulzenbacher et al., J. Mol. Biol. 342(2):489-502 (2004)).

Another pathway for converting pyruvate to isobutanol comprises thefollowing substrate to product conversions (FIG. 1, steps a, b, c, f, g,e):

-   -   a) pyruvate to acetolactate, as catalyzed for example by        acetolactate synthase,    -   b) acetolactate to 2,3-dihydroxyisovalerate, as catalyzed for        example by acetohydroxy acid isomeroreductase,    -   c) 2,3-dihydroxyisovalerate to α-ketoisovalerate, as catalyzed        for example by acetohydroxy acid dehydratase,    -   f) α-ketoisovalerate to isobutyryl-CoA, as catalyzed for example        by a branched-chain keto acid dehydrogenase,    -   g) isobutyryl-CoA to isobutyraldehyde, as catalyzed for example        by an acylating aldehyde dehydrogenase, and    -   e) isobutyraldehyde to isobutanol, as catalyzed for example by,        a branched-chain alcohol dehydrogenase.

The first three steps in this pathway (a, b, c) are the same as thosedescribed above. The α-ketoisovalerate is converted to isobutyryl-CoA bythe action of a branched-chain keto acid dehydrogenase. While yeast canonly use valine as a nitrogen source, many other organisms (botheukaryotes and prokaryotes) can use valine as the carbon source as well.These organisms have branched-chain keto acid dehydrogenase (Sokatch etal. J. Bacteriol. 148(2):647-652 (1981)), which generatesisobutyryl-CoA. Isobutyryl-CoA may be converted to isobutyraldehyde byan acylating aldehyde dehydrogenase. Dehydrogenases active with thebranched-chain substrate have been described, but not cloned, inLeuconostoc and Propionibacterium (Kazahaya et al., J. Gen. Appl.Microbiol. 18:43-55 (1972); Hosoi et al., J. Ferment. Technol.57:418-427 (1979)). However, it is also possible that acylating aldehydedehydrogenases known to function with straight-chain acyl-CoAs (i.e.butyryl-CoA), may also work with isobutyryl-CoA. The isobutyraldehyde isthen converted to isobutanol by a branched-chain alcohol dehydrogenase,as described above for the first pathway.

Another pathway for converting pyruvate to isobutanol comprises thefollowing substrate to product conversions (FIG. 1, steps a, b, c, h, i,j, e):

-   -   a) pyruvate to acetolactate, as catalyzed for example by        acetolactate synthase,    -   b) acetolactate to 2,3-dihydroxyisovalerate, as catalyzed for        example by acetohydroxy acid isomeroreductase,    -   c) 2,3-dihydroxyisovalerate to α-ketoisovalerate, as catalyzed        for example by acetohydroxy acid dehydratase,    -   h) α-ketoisovalerate to valine, as catalyzed for example by        valine dehydrogenase or transaminase,    -   i) valine to isobutylamine, as catalyzed for example by valine        decarboxylase,    -   j) isobutylamine to isobutyraldehyde, as catalyzed for example        by omega transaminase, and    -   e) isobutyraldehyde to isobutanol, as catalyzed for example by,        a branched-chain alcohol dehydrogenase.

The first three steps in this pathway (a, b, c) are the same as thosedescribed above. This pathway requires the addition of a valinedehydrogenase or a suitable transaminase. Valine (and or leucine)dehydrogenase catalyzes reductive amination and uses ammonia; K_(m)values for ammonia are in the millimolar range (Priestly et al., BiochemJ. 261(3):853-861 (1989); Vancura et al., J. Gen. Microbiol.134(12):3213-3219 (1988) Zink et al., Arch. Biochem. Biophys. 99:72-77(1962); Sekimoto et al. J. Biochem (Japan) 116(1):176-182 (1994)).Transaminases typically use either glutamate or alanine as amino donorsand have been characterized from a number of organisms (Lee-Peng et al.,J. Bacteriol. 139(2):339-345 (1979); Berg et al., J. Bacteriol.155(3):1009-1014 (1983)). An alanine-specific enzyme may be desirable,since the generation of pyruvate from this step could be coupled to theconsumption of pyruvate later in the pathway when the amine group isremoved (see below). The next step is decarboxylation of valine, areaction that occurs in valanimycin biosynthesis in Streptomyces (Garget al., Mol. Microbiol. 46(2):505-517 (2002)). The resultingisobutylamine may be converted to isobutyraldehyde in a pyridoxal5′-phosphate-dependent reaction by, for example, an enzyme of theomega-aminotransferase family. Such an enzyme from Vibrio fluvialis hasdemonstrated activity with isobutylamine (Shin et al., Biotechnol.Bioeng. 65(2):206-211 (1999)). Another omega-aminotransferase fromAlcaligenes denitrificans has been cloned and has some activity withbutylamine (Yun et al., Appl. Environ. Microbiol. 70(4):2529-2534(2004)). In this direction, these enzymes use pyruvate as the aminoacceptor, yielding alanine. As mentioned above, adverse affects on thepyruvate pool may be offset by using a pyruvate-producing transaminaseearlier in the pathway. The isobutyraldehyde is then converted toisobutanol by a branched-chain alcohol dehydrogenase, as described abovefor the first pathway.

The fourth isobutanol biosynthetic pathway comprises the substrate toproduct conversions shown as steps k, g, e in FIG. 1. A number oforganisms are known to produce butyrate and/or butanol via a butyryl-CoAintermediate (Durre et al., FEMS Microbiol. Rev. 17(3):251-262 (1995);Abbad-Andaloussi et al., Microbiology 142(5):1149-1158 (1996)).Isobutanol production may be engineered in these organisms by additionof a mutase able to convert butyryl-CoA to isobutyryl-CoA (FIG. 1, stepk). Genes for both subunits of isobutyryl-CoA mutase, a coenzymeB₁₂-dependent enzyme, have been cloned from a Streptomycete(Ratnatilleke et al., J. Biol. Chem. 274(44):31679-31685 (1999)). Theisobutyryl-CoA is converted to isobutyraldehyde (step g in FIG. 1),which is converted to isobutanol (step e in FIG. 1).

Thus, in providing multiple recombinant pathways from pyruvate toisobutanol, there exist a number of choices to fulfill the individualconversion steps, and the person of skill in the art will be able toutilize publicly available sequences to construct the relevant pathways.A listing of a representative number of genes known in the art anduseful in the construction of isobutanol biosynthetic pathways arelisted below in Table 2.

TABLE 2 Sources of Isobutanol Biosynthetic Pathway Genes Gene GenBankCitation acetolactate synthase Z99122, Bacillus subtilis complete genome(section 19 of 21): from 3608981 to 3809670gi|32468830|emb|Z99122.2|BSUB0019[32468830] M73842, Klebsiellapneumoniae acetolactate synthase (iluk) gene, complete cdsgi|149210|gb|M73842.1|KPNILUK[149210] L16975, Lactococcus lactisalpha-acetolactate synthase (als) gene, complete cdsgi|473900|gb|L16975.1|LACALS[473900] acetohydroxy acid NC_000913,Escherichia coli K12, complete genome isomeroreductasegi|49175990|ref|NC_000913.2|[49175990] NC_001144, Saccharomycescerevisiae chromosome XII, complete chromosome sequencegi|42742286|ref|NC_001144.3|[42742286] BX957220, Methanococcusmaripaludis S2 complete genome; segment 2/5gi|44920669|emb|BX957220.1|[44920669] Z99118, Bacillus subtilis completegenome (section 15 of 21): from 2812801 to 3013507gi|32468802|emb|Z99118.2|BSUB0015[32468802] acetohydroxy acid NC_000913,Escherichia coli K12, complete genome dehydratasegi|49175990|ref|NC_000913.2|[49175990] NC_001142, Saccharomycescerevisiae chromosome X, complete chromosome sequencegi|42742252|ref|NC_001142.5|[42742252] BX957219, Methanococcusmaripaludis S2 complete genome; segment 1/5gi|45047123|emb|BX957219.1|[45047123] Z99115, Bacillus subtilis completegenome (section 12 of 21): from 2207806 to 2409180gi|32468778|emb|Z99115.2|BSUB0012[32468778] branched-chain α-ketoAY548760, Lactococcus lactis branched-chain alpha- acid decarboxylaseketoacid decarboxylase (kdcA) gene, complete cdsgi|44921616|gb|AY548760.1|[44921616] AJ746364, Lactococcus lactis subsp.lactis kivd gene for alpha-ketoisovalerate decarboxylase, strain IFPL730gi|51870501|emb|AJ746364.1|[51870501] NC_003197, Salmonella typhimuriumLT2, complete genome gi|16763390|ref|NC_003197.1|[16763390] NC_001988,Clostridium acetobutylicum ATCC 824 plasmid pSOL1, complete sequencegi|15004705|ref|NC_001988.2|[15004705] branched-chain NC_001136,Saccharomyces cerevisiae chromosome alcohol IV, complete chromosomesequence dehydrogenase gi|50593138|ref|NC_001136.6|[50593138] NC_001145,Saccharomyces cerevisiae chromosome XIII, complete chromosome sequencegi|44829554|ref|NC_001145.2|[44829554] NC_000913, Escherichia coli K12,complete genome gi|49175990|ref|NC_000913.2|[49175990] NC_003030,Clostridium acetobutylicum ATCC 824, complete genomegi|15893298|ref|NC_003030.1|[15893298] branched-chain keto Z99116,Bacillus subtilis complete genome (section 13 acid dehydrogenase of 21):from 2409151 to 2613687 gi|32468787|emb|Z99116.2|BSUB0013[32468787]M57613, Pseudomonas putida branched-chain keto acid dehydrogenase operon(bkdA1, bkdA1 and bkdA2), transacylase E2 (bkdB), bkdR and lipoamidedehydrogenase (lpdV) genes, complete cdsgi|790512|gb|M57613.1|PSEBKDPPG2[790512] acylating aldehyde AF157306,Clostridium beijerinckii strain NRRL B593 dehydrogenase hypotheticalprotein, coenzyme A acylating aldehyde dehydrogenase (ald),acetoacetate:butyrate/acetate coenzyme A transferase (ctfA),acetoacetate:butyrate/acetate coenzyme A transferase (ctfB), andacetoacetate decarboxylase (adc) genes, complete cdsgi|47422980|gb|AF157306.2|[47422980] NC_001988, Clostridiumacetobutylicum ATCC 824 plasmid pSOL1, complete sequencegi|15004705|ref|NC_001988.2|[15004705] U13232, Pseudomonas putidaNCIB9816 acetaldehyde dehydrogenase (nahO) and 4-hydroxy-2-oxovaleratealdolase (nahM) genes, complete cds, and 4- oxalocrotonate decarboxylase(nahK) and 2-oxopent-4- enoate hydratase (nahL) genes, partial cdsgi|595671|gb|U13232.1|PPU13232[595671] transaminase NC_000913,Escherichia coli K12, complete genomegi|49175990|ref|NC_000913.2|[49175990] NC_006322, Bacillus licheniformisATCC 14580, complete genome gi|52783855|ref|NC_006322.1|[52783855]NC_001142, Saccharomyces cerevisiae chromosome X, complete chromosomesequence gi|42742252|ref|NC_001142.5|[42742252] NC_000916,Methanothermobacter thermautotrophicus str. Delta H, complete genomegi|15678031|ref|NC_000916.1|[15678031] valine dehydrogenase NC_003888,Streptomyces coelicolor A3(2), complete genomegi|32141095|ref|NC_003888.3|[32141095] Z99116, Bacillus subtiliscomplete genome (section 13 of 21): from 2409151 to 2613687gi|32468787|emb|Z99116.2|BSUB0013[32468787] valine decarboxylaseAY116644, Streptomyces viridifaciens amino acid aminotransferase gene,partial cds; ketol-acid reductoisomerase, acetolactate synthetase smallsubunit, acetolactate synthetase large subunit, complete cds; azoxyantibiotic valanimycin gene cluster, complete sequence; and putativetransferase, and putative secreted protein genes, complete cdsgi|27777548|gb|AY116644.1|[27777548] omega transaminase AY330220,Achromobacter denitrificans omega-amino acid:pyruvate transaminase(aptA) gene, complete cds gi|33086797|gb|AY330220.1|[33086797]NC_007347, Ralstonia eutropha JMP134 chromosome 1, complete sequencegi|73539706|ref|NC_007347.1|[73539706] NC_004347, Shewanella oneidensisMR-1, complete genome gi|24371600|ref|NC_004347.1|[24371600]NZ_AAAG02000002, Rhodospirillum rubrum Rrub02_2, whole genome shotgunsequence gi|48764549|ref|NZ_AAAG02000002.1|[48764549] AE016776,Pseudomonas putida KT2440 section 3 of 21 of the complete genomegi|26557019|gb|AE016776.1|[26557019] isobutyryl-CoA mutase U67612,Streptomyces cinnamonensis coenzyme B12- dependent isobutyrylCoA mutase(icm) gene, complete cds gi|3002491|gb|U67612.1|SCU67612[3002491]AJ246005, Streptomyces cinnamonensis icmB gene for isobutyryl-CoAmutase, small subunit gi|6137076|emb|AJ246005.1|SCI246005[6137076]AL939123, Streptomyces coelicolor A3(2) complete genome; segment 20/29gi|24430032|emb|AL939123.1|SCO939123[24430032] AL9939121, Streptomycescoelicolor A3(2) complete genome; segment 18/29gi|24429533|emb|AL939121.1|SCO939121[24429533] NC_003155, Streptomycesavermitilis MA-4680, complete genomegi|57833846|ref|NC_003155.3|[57833846]Microbial Hosts for Isobutanol Production

Microbial hosts for isobutanol production may be selected from bacteria,cyanobacteria, filamentous fungi and yeasts. The microbial host used forisobutanol production is preferably tolerant to isobutanol so that theyield is not limited by butanol toxicity. Microbes that aremetabolically active at high titer levels of isobutanol are not wellknown in the art. Although butanol-tolerant mutants have been isolatedfrom solventogenic Clostridia, little information is availableconcerning the butanol tolerance of other potentially useful bacterialstrains. Most of the studies on the comparison of alcohol tolerance inbacteria suggest that butanol is more toxic than ethanol (de Cavalho etal., Microsc. Res. Tech. 64:215-22 (2004) and Kabelitz et al., FEMSMicrobiol. Lett. 220:223-227 (2003)). Tomas et al. (J. Bacteriol.186:2006-2018 (2004)) report that the yield of 1-butanol duringfermentation in Clostridium acetobutylicum may be limited by 1-butanoltoxicity. The primary effect of 1-butanol on Clostridium acetobutylicumis disruption of membrane functions (Hermann et al., Appl. Environ.Microbiol. 50:1238-1243 (1985)).

The microbial hosts selected for the production of isobutanol arepreferably tolerant to isobutanol and should be able to convertcarbohydrates to isobutanol. The criteria for selection of suitablemicrobial hosts include the following: intrinsic tolerance toisobutanol, high rate of glucose utilization, availability of genetictools for gene manipulation, and the ability to generate stablechromosomal alterations.

Suitable host strains with a tolerance for isobutanol may be identifiedby screening based on the intrinsic tolerance of the strain. Theintrinsic tolerance of microbes to isobutanol may be measured bydetermining the concentration of isobutanol that is responsible for 50%inhibition of the growth rate (IC50) when grown in a minimal medium. TheIC50 values may be determined using methods known in the art. Forexample, the microbes of interest may be grown in the presence ofvarious amounts of isobutanol and the growth rate monitored by measuringthe optical density at 600 nanometers. The doubling time may becalculated from the logarithmic part of the growth curve and used as ameasure of the growth rate. The concentration of isobutanol thatproduces 50% inhibition of growth may be determined from a graph of thepercent inhibition of growth versus the isobutanol concentration.Preferably, the host strain should have an IC50 for isobutanol ofgreater than about 0.5%.

The microbial host for isobutanol production should also utilize glucoseat a high rate. Most microbes are capable of utilizing carbohydrates.However, certain environmental microbes cannot utilize carbohydrates tohigh efficiency, and therefore would not be suitable hosts.

The ability to genetically modify the host is essential for theproduction of any recombinant microorganism. The mode of gene transfertechnology may be by electroporation, conjugation, transduction ornatural transformation. A broad range of host conjugative plasmids anddrug resistance markers are available. The cloning vectors are tailoredto the host organisms based on the nature of antibiotic resistancemarkers that can function in that host.

The microbial host also has to be manipulated in order to inactivatecompeting pathways for carbon flow by deleting various genes. Thisrequires the availability of either transposons to direct inactivationor chromosomal integration vectors. Additionally, the production hostshould be amenable to chemical mutagenesis so that mutations to improveintrinsic isobutanol tolerance may be obtained.

Based on the criteria described above, suitable microbial hosts for theproduction of isobutanol include, but are not limited to, members of thegenera Clostridium, Zymomonas, Escherichia, Salmonella, Rhodococcus,Pseudomonas, Bacillus, Lactobacillus, Enterococcus, Alcaligenes,Klebsiella, Paenibacillus, Arthrobacter, Corynebacterium,Brevibacterium, Pichia, Candida, Hansenula and Saccharomyces. Preferredhosts include: Escherichia coli, Alcaligenes eutrophus, Bacilluslicheniformis, Paenibacillus macerans, Rhodococcus erythropolis,Pseudomonas putida, Lactobacillus plantarum, Enterococcus faecium,Enterococcus gallinarium, Enterococcus faecalis, Bacillus subtilis andSaccharomyces cerevisiae.

Construction of Production Host

Recombinant organisms containing the necessary genes that will encodethe enzymatic pathway for the conversion of a fermentable carbonsubstrate to isobutanol may be constructed using techniques well knownin the art. In the present invention, genes encoding the enzymes of oneof the isobutanol biosynthetic pathways of the invention, for example,acetolactate synthase, acetohydroxy acid isomeroreductase, acetohydroxyacid dehydratase, branched-chain α-keto acid decarboxylase, andbranched-chain alcohol dehydrogenase, may be isolated from varioussources, as described above.

Methods of obtaining desired genes from a bacterial genome are commonand well known in the art of molecular biology. For example, if thesequence of the gene is known, suitable genomic libraries may be createdby restriction endonuclease digestion and may be screened with probescomplementary to the desired gene sequence. Once the sequence isisolated, the DNA may be amplified using standard primer-directedamplification methods such as polymerase chain reaction (U.S. Pat. No.4,683,202) to obtain amounts of DNA suitable for transformation usingappropriate vectors. Tools for codon optimization for expression in aheterologous host are readily available. Some tools for codonoptimization are available based on the GC content of the host organism.The GC content of some exemplary microbial hosts is given Table 3.

TABLE 3 GC Content of Microbial Hosts Strain % GC B. licheniformis 46 B.subtilis 42 C. acetobutylicum 37 E. coli 50 P. putida 61 A. eutrophus 61Paenibacillus macerans 51 Rhodococcus erythropolis 62 Brevibacillus 50Paenibacillus polymyxa 50

Once the relevant pathway genes are identified and isolated they may betransformed into suitable expression hosts by means well known in theart. Vectors or cassettes useful for the transformation of a variety ofhost cells are common and commercially available from companies such asEPICENTRE® (Madison, Wis.), Invitrogen Corp. (Carlsbad, Calif.),Stratagene (La Jolla, Calif.), and New England Biolabs, Inc. (Beverly,Mass.). Typically the vector or cassette contains sequences directingtranscription and translation of the relevant gene, a selectable marker,and sequences allowing autonomous replication or chromosomalintegration. Suitable vectors comprise a region 5′ of the gene whichharbors transcriptional initiation controls and a region 3′ of the DNAfragment which controls transcriptional termination. Both controlregions may be derived from genes homologous to the transformed hostcell, although it is to be understood that such control regions may alsobe derived from genes that are not native to the specific species chosenas a production host.

Initiation control regions or promoters, which are useful to driveexpression of the relevant pathway coding regions in the desired hostcell are numerous and familiar to those skilled in the art. Virtuallyany promoter capable of driving these genetic elements is suitable forthe present invention including, but not limited to, CYC1, HIS3, GAL1,GAL10, ADH1, PGK, PHO5, GAPDH, ADC1, TRP1, URA3, LEU2, ENO, TPI, CUP1,FBA, GPD, and GPM (useful for expression in Saccharomyces); AOX1 (usefulfor expression in Pichia); and lac, ara, tet, trp, IP_(L), IP_(R), T7,tac, and trc (useful for expression in Escherichia coli, Alcaligenes,and Pseudomonas); the amy, apr, npr promoters and various phagepromoters useful for expression in Bacillus subtilis, Bacilluslicheniformis, and Paenibacillus macerans; nisA (useful for expressionGram-positive bacteria, Eichenbaum et al. Appl. Environ. Microbiol.64(8):2763-2769 (1998)); and the synthetic P11 promoter (useful forexpression in Lactobacillus plantarum, Rud et al., Microbiology152:1011-1019 (2006)).

Termination control regions may also be derived from various genesnative to the preferred hosts. Optionally, a termination site may beunnecessary, however, it is most preferred if included.

Certain vectors are capable of replicating in a broad range of hostbacteria and can be transferred by conjugation. The complete andannotated sequence of pRK404 and three related vectors-pRK437, pRK442,and pRK442(H) are available. These derivatives have proven to bevaluable tools for genetic manipulation in Gram-negative bacteria (Scottet al., Plasmid 50(1):74-79 (2003)). Several plasmid derivatives ofbroad-host-range Inc P4 plasmid RSF1010 are also available withpromoters that can function in a range of Gram-negative bacteria.Plasmid pAYC36 and pAYC37, have active promoters along with multiplecloning sites to allow for the heterologous gene expression inGram-negative bacteria.

Chromosomal gene replacement tools are also widely available. Forexample, a thermosensitive variant of the broad-host-range repliconpWV101 has been modified to construct a plasmid pVE6002 which can beused to effect gene replacement in a range of Gram-positive bacteria(Maguin et al., J. Bacteriol. 174(17):5633-5638 (1992)). Additionally,in vitro transposomes are available to create random mutations in avariety of genomes from commercial sources such as EPICENTRE®.

The expression of an isobutanol biosynthetic pathway in variouspreferred microbial hosts is described in more detail below.

Expression of an Isobutanol Biosynthetic Pathway in E. coli

Vectors or cassettes useful for the transformation of E. coli are commonand commercially available from the companies listed above. For example,the genes of an isobutanol biosynthetic pathway may be isolated fromvarious sources, cloned into a modified pUC19 vector and transformedinto E. coli NM522, as described in Examples 6 and 7.

Expression of an Isobutanol Biosynthetic Pathway in Rhodococcuserythropolis

A series of E. coli-Rhodococcus shuttle vectors are available forexpression in R. erythropolis, including, but not limited to, pRhBR17and pDA71 (Kostichka et al., Appl. Microbiol. Biotechnol. 62:61-68(2003)). Additionally, a series of promoters are available forheterologous gene expression in R. erythropolis (see for exampleNakashima et al., Appl. Environ. Microbiol. 70:5557-5568 (2004), and Taoet al., Appl. Microbiol. Biotechnol. 2005, DOI 10.1007/s00253-005-0064).Targeted gene disruption of chromosomal genes in R. erythropolis may becreated using the method described by Tao et al., supra, and Brans etal. (Appl. Environ. Microbiol. 66: 2029-2036 (2000)).

The heterologous genes required for the production of isobutanol, asdescribed above, may be cloned initially in pDA71 or pRhBR71 andtransformed into E. coli. The vectors may then be transformed into R.erythropolis by electroporation, as described by Kostichka et al.,supra. The recombinants may be grown in synthetic medium containingglucose and the production of isobutanol can be followed using methodsknown in the art.

Expression of an Isobutanol Biosynthetic Pathway in B. Subtilis

Methods for gene expression and creation of mutations in B. subtilis arealso well known in the art. For example, the genes of an isobutanolbiosynthetic pathway may be isolated from various sources, cloned into amodified pUC19 vector and transformed into Bacillus subtilis BE1010, asdescribed in Example 8. Additionally, the five genes of an isobutanolbiosynthetic pathway can be split into two operons for expression, asdescribed in Example 20. The three genes of the pathway (bubB, ilvD, andkivD) were integrated into the chromosome of Bacillus subtilis BE1010(Payne and Jackson, J. Bacteriol. 173:2278-2282 (1991)). The remainingtwo genes (ilvC and bdhB) were cloned into an expression vector andtransformed into the Bacillus strain carrying the integrated isobutanolgenes

Expression of an Isobutanol Biosynthetic Pathway in B. Licheniformis

Most of the plasmids and shuttle vectors that replicate in B. subtilismay be used to transform B. licheniformis by either protoplasttransformation or electroporation. The genes required for the productionof isobutanol may be cloned in plasmids pBE20 or pBE60 derivatives(Nagarajan et al., Gene 114:121-126 (1992)). Methods to transform B.licheniformis are known in the art (for example see Fleming et al. Appl.Environ. Microbiol., 61(11):3775-3780 (1995)). The plasmids constructedfor expression in B. subtilis may be transformed into B. licheniformisto produce a recombinant microbial host that produces isobutanol.

Expression of an Isobutanol Biosynthetic Pathway in Paenibacillusmacerans

Plasmids may be constructed as described above for expression in B.subtilis and used to transform Paenibacillus macerans by protoplasttransformation to produce a recombinant microbial host that producesisobutanol.

Expression of the isobutanol biosynthetic pathway in Alcaligenes(Ralstonia) eutrophus

Methods for gene expression and creation of mutations in Alcaligeneseutrophus are known in the art (see for example Taghavi et al., Appl.Environ. Microbiol., 60(10):3585-3591 (1994)). The genes for anisobutanol biosynthetic pathway may be cloned in any of the broad hostrange vectors described above, and electroporated to generaterecombinants that produce isobutanol. The poly(hydroxybutyrate) pathwayin Alcaligenes has been described in detail, a variety of genetictechniques to modify the Alcaligenes eutrophus genome is known, andthose tools can be applied for engineering an isobutanol biosyntheticpathway.

Expression of an Isobutanol Biosynthetic Pathway in Pseudomonas putida

Methods for gene expression in Pseudomonas putida are known in the art(see for example Ben-Bassat et al., U.S. Pat. No. 6,586,229, which isincorporated herein by reference). The butanol pathway genes may beinserted into pPCU18 and this ligated DNA may be electroporated intoelectrocompetent Pseudomonas putida DOT-T1 C5aAR1 cells to generaterecombinants that produce isobutanol.

Expression of an Isobutanol Biosynthetic Pathway in Saccharomycescerevisiae

Methods for gene expression in Saccharomyces cerevisiae are known in theart (see for example Methods in Enzymology, Volume 194, Guide to YeastGenetics and Molecular and Cell Biology (Part A, 2004, Christine Guthrieand Gerald R. Fink (Eds.), Elsevier Academic Press, San Diego, Calif.).Expression of genes in yeast typically requires a promoter, followed bythe gene of interest, and a transcriptional terminator. A number ofyeast promoters can be used in constructing expression cassettes forgenes encoding an isobutanol biosynthetic pathway, including, but notlimited to constitutive promoters FBA, GPD, ADH1, and GPM, and theinducible promoters GAL1, GAL10, and CUP1. Suitable transcriptionalterminators include, but are not limited to FBAt, GPDt, GPMt, ERG10t,GAL1t, CYC1, and ADH1. For example, suitable promoters, transcriptionalterminators, and the genes of an isobutanol biosynthetic pathway may becloned into E. coli-yeast shuttle vectors as described in Example 17.

Expression of an Isobutanol Biosynthetic Pathway in Lactobacillusplantarum

The Lactobacillus genus belongs to the Lactobacillales family and manyplasmids and vectors used in the transformation of Bacillus subtilis andStreptococcus may be used for lactobacillus. Non-limiting examples ofsuitable vectors include pAMβ1 and derivatives thereof (Renault et al.,Gene 183:175-182 (1996); and O'Sullivan et al., Gene 137:227-231(1993)); pMBB1 and pHW800, a derivative of pMBB1 (Wyckoff et al. Appl.Environ. Microbiol. 62:1481-1486 (1996)); pMG1, a conjugative plasmid(Tanimoto et al., J. Bacteriol. 184:5800-5804 (2002)); pNZ9520(Kleerebezem et al., Appl. Environ. Microbiol. 63:4581-4584 (1997));pAM401 (Fujimoto et al., Appl. Environ. Microbiol. 67:1262-1267 (2001));and pAT392 (Arthur et al., Antimicrob. Agents Chemother. 38:1899-1903(1994)). Several plasmids from Lactobacillus plantarum have also beenreported (e.g., van Kranenburg R, Golic N, Bongers R, Leer R J, de Vos WM, Siezen R J, Kleerebezem M. Appl. Environ. Microbiol. 2005 March;71(3): 1223-1230). For example, expression of an isobutanol biosyntheticpathway in Lactobacillus plantarum is described in Example 21.

Expression of an Isobutanol Biosynthetic Pathway in Enterococcusfaecium, Enterococcus gallinarium, and Enterococcus faecalis

The Enterococcus genus belongs to the Lactobacillales family and manyplasmids and vectors used in the transformation of Lactobacillus,Bacillus subtilis, and Streptococcus may be used for Enterococcus.Non-limiting examples of suitable vectors include pAMβ1 and derivativesthereof (Renault et al., Gene 183:175-182 (1996); and O'Sullivan et al.,Gene 137:227-231 (1993)); pMBB1 and pHW800, a derivative of pMBB1(Wyckoff et al. Appl. Environ. Microbiol. 62:1481-1486 (1996)); pMG1, aconjugative plasmid (Tanimoto et al., J. Bacteriol. 184:5800-5804(2002)); pNZ9520 (Kleerebezem et al., Appl. Environ. Microbiol.63:4581-4584 (1997)); pAM401 (Fujimoto et al., Appl. Environ. Microbiol.67:1262-1267 (2001)); and pAT392 (Arthur et al., Antimicrob. AgentsChemother. 38:1899-1903 (1994)). Expression vectors for E. faecalisusing the nisA gene from Lactococcus may also be used (Eichenbaum etal., Appl. Environ. Microbiol. 64:2763-2769 (1998). Additionally,vectors for gene replacement in the E. faecium chromosome may be used(Nallaapareddy et al., Appl. Environ. Microbiol. 72:334-345 (2006)). Forexample, expression of an isobutanol biosynthetic pathway inEnterococcus faecalis is described in Example 22.

Fermentation Media

Fermentation media in the present invention must contain suitable carbonsubstrates. Suitable substrates may include, but are not limited to,monosaccharides such as glucose and fructose, oligosaccharides such aslactose or sucrose, polysaccharides such as starch or cellulose ormixtures thereof and unpurified mixtures from renewable feedstocks suchas cheese whey permeate, cornsteep liquor, sugar beet molasses, andbarley malt. Additionally the carbon substrate may also be one-carbonsubstrates such as carbon dioxide, or methanol for which metabolicconversion into key biochemical intermediates has been demonstrated. Inaddition to one and two carbon substrates methylotrophic organisms arealso known to utilize a number of other carbon containing compounds suchas methylamine, glucosamine and a variety of amino acids for metabolicactivity. For example, methylotrophic yeast are known to utilize thecarbon from methylamine to form trehalose or glycerol (Bellion et al.,Microb. Growth C1 Compd., [Int. Symp.], 7th (1993), 415-32. Editor(s):Murrell, J. Collin; Kelly, Don P. Publisher: Intercept, Andover, UK).Similarly, various species of Candida will metabolize alanine or oleicacid (Sulter et al., Arch. Microbiol. 153:485-489 (1990)). Hence it iscontemplated that the source of carbon utilized in the present inventionmay encompass a wide variety of carbon containing substrates and willonly be limited by the choice of organism.

Although it is contemplated that all of the above mentioned carbonsubstrates and mixtures thereof are suitable in the present invention,preferred carbon substrates are glucose, fructose, and sucrose.

In addition to an appropriate carbon source, fermentation media mustcontain suitable minerals, salts, cofactors, buffers and othercomponents, known to those skilled in the art, suitable for the growthof the cultures and promotion of the enzymatic pathway necessary forisobutanol production.

Culture Conditions

Typically cells are grown at a temperature in the range of about 25° C.to about 40° C. in an appropriate medium. Suitable growth media in thepresent invention are common commercially prepared media such as LuriaBertani (LB) broth, Sabouraud Dextrose (SD) broth or Yeast medium (YM)broth. Other defined or synthetic growth media may also be used, and theappropriate medium for growth of the particular microorganism will beknown by one skilled in the art of microbiology or fermentation science.The use of agents known to modulate catabolite repression directly orindirectly, e.g., cyclic adenosine 2′:3′-monophosphate, may also beincorporated into the fermentation medium.

Suitable pH ranges for the fermentation are between pH 5.0 to pH 9.0,where pH 6.0 to pH 8.0 is preferred as the initial condition.

Fermentations may be performed under aerobic or anaerobic conditions,where anaerobic or microaerobic conditions are preferred.

The amount of isobutanol produced in the fermentation medium can bedetermined using a number of methods known in the art, for example, highperformance liquid chromatography (HPLC) or gas chromatography (GC).

Industrial Batch and Continuous Fermentations

The present process employs a batch method of fermentation. A classicalbatch fermentation is a closed system where the composition of themedium is set at the beginning of the fermentation and not subject toartificial alterations during the fermentation. Thus, at the beginningof the fermentation the medium is inoculated with the desired organismor organisms, and fermentation is permitted to occur without addinganything to the system. Typically, however, a “batch” fermentation isbatch with respect to the addition of carbon source and attempts areoften made at controlling factors such as pH and oxygen concentration.In batch systems the metabolite and biomass compositions of the systemchange constantly up to the time the fermentation is stopped. Withinbatch cultures cells moderate through a static lag phase to a highgrowth log phase and finally to a stationary phase where growth rate isdiminished or halted. If untreated, cells in the stationary phase willeventually die. Cells in log phase generally are responsible for thebulk of production of end product or intermediate.

A variation on the standard batch system is the Fed-Batch system.Fed-Batch fermentation processes are also suitable in the presentinvention and comprise a typical batch system with the exception thatthe substrate is added in increments as the fermentation progresses.Fed-Batch systems are useful when catabolite repression is apt toinhibit the metabolism of the cells and where it is desirable to havelimited amounts of substrate in the media. Measurement of the actualsubstrate concentration in Fed-Batch systems is difficult and istherefore estimated on the basis of the changes of measurable factorssuch as pH, dissolved oxygen and the partial pressure of waste gasessuch as CO₂. Batch and Fed-Batch fermentations are common and well knownin the art and examples may be found in Thomas D. Brock inBiotechnology: A Textbook of Industrial Microbiology, Second Edition(1989) Sinauer Associates, Inc., Sunderland, Mass., or Deshpande, MukundV., Appl. Biochem. Biotechnol., 36:227, (1992), herein incorporated byreference.

Although the present invention is performed in batch mode it iscontemplated that the method would be adaptable to continuousfermentation methods. Continuous fermentation is an open system where adefined fermentation medium is added continuously to a bioreactor and anequal amount of conditioned media is removed simultaneously forprocessing. Continuous fermentation generally maintains the cultures ata constant high density where cells are primarily in log phase growth.

Continuous fermentation allows for the modulation of one factor or anynumber of factors that affect cell growth or end product concentration.For example, one method will maintain a limiting nutrient such as thecarbon source or nitrogen level at a fixed rate and allow all otherparameters to moderate. In other systems a number of factors affectinggrowth can be altered continuously while the cell concentration,measured by media turbidity, is kept constant. Continuous systems striveto maintain steady state growth conditions and thus the cell loss due tothe medium being drawn off must be balanced against the cell growth ratein the fermentation. Methods of modulating nutrients and growth factorsfor continuous fermentation processes as well as techniques formaximizing the rate of product formation are well known in the art ofindustrial microbiology and a variety of methods are detailed by Brock,supra.

It is contemplated that the present invention may be practiced usingeither batch, fed-batch or continuous processes and that any known modeof fermentation would be suitable. Additionally, it is contemplated thatcells may be immobilized on a substrate as whole cell catalysts andsubjected to fermentation conditions for isobutanol production.

Methods for Isobutanol Isolation from the Fermentation Medium

The bioproduced isobutanol may be isolated from the fermentation mediumusing methods known in the art. For example, solids may be removed fromthe fermentation medium by centrifugation, filtration, decantation, orthe like. Then, the isobutanol may be isolated from the fermentationmedium, which has been treated to remove solids as described above,using methods such as distillation, liquid-liquid extraction, ormembrane-based separation. Because isobutanol forms a low boiling point,azeotropic mixture with water, distillation can only be used to separatethe mixture up to its azeotropic composition. Distillation may be usedin combination with another separation method to obtain separationaround the azeotrope. Methods that may be used in combination withdistillation to isolate and purify isobutanol include, but are notlimited to, decantation, liquid-liquid extraction, adsorption, andmembrane-based techniques. Additionally, isobutanol may be isolatedusing azeotropic distillation using an entrainer (see for exampleDoherty and Malone, Conceptual Design of Distillation Systems, McGrawHill, New York, 2001).

The isobutanol-water mixture forms a heterogeneous azeotrope so thatdistillation may be used in combination with decantation to isolate andpurify the isobutanol. In this method, the isobutanol containingfermentation broth is distilled to near the azeotropic composition.Then, the azeotropic mixture is condensed, and the isobutanol isseparated from the fermentation medium by decantation. The decantedaqueous phase may be returned to the first distillation column asreflux. The isobutanol-rich decanted organic phase may be furtherpurified by distillation in a second distillation column.

The isobutanol may also be isolated from the fermentation medium usingliquid-liquid extraction in combination with distillation. In thismethod, the isobutanol is extracted from the fermentation broth usingliquid-liquid extraction with a suitable solvent. Theisobutanol-containing organic phase is then distilled to separate theisobutanol from the solvent.

Distillation in combination with adsorption may also be used to isolateisobutanol from the fermentation medium. In this method, thefermentation broth containing the isobutanol is distilled to near theazeotropic composition and then the remaining water is removed by use ofan adsorbent, such as molecular sieves (Aden et al. LignocellulosicBiomass to Ethanol Process Design and Economics Utilizing Co-CurrentDilute Acid Prehydrolysis and Enzymatic Hydrolysis for Corn Stover,Report NREL/TP-510-32438, National Renewable Energy Laboratory, June2002).

Additionally, distillation in combination with pervaporation may be usedto isolate and purify the isobutanol from the fermentation medium. Inthis method, the fermentation broth containing the isobutanol isdistilled to near the azeotropic composition, and then the remainingwater is removed by pervaporation through a hydrophilic membrane (Guo etal., J. Membr. Sci. 245, 199-210 (2004)).

EXAMPLES

The present invention is further defined in the following Examples. Itshould be understood that these Examples, while indicating preferredembodiments of the invention, are given by way of illustration only.From the above discussion and these Examples, one skilled in the art canascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various uses andconditions.

General Methods

Standard recombinant DNA and molecular cloning techniques used in theExamples are well known in the art and are described by Sambrook, J.,Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual;Cold Spring Harbor Laboratory Press: Cold Spring Harbor, N.Y. (1989)(Maniatis) and by T. J. Silhavy, M. L. Bennan, and L. W. Enquist,Experiments with Gene Fusions, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1984) and by Ausubel, F. M. et al., CurrentProtocols in Molecular Biology, pub. by Greene Publishing Assoc. andWiley-Interscience (1987).

Materials and methods suitable for the maintenance and growth ofbacterial cultures are well known in the art. Techniques suitable foruse in the following Examples may be found as set out in Manual ofMethods for General Bacteriology (Phillipp Gerhardt, R. G. E. Murray,Ralph N. Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg andG. Briggs Phillips, eds), American Society for Microbiology, Washington,D.C. (1994)) or by Thomas D. Brock in Biotechnology: A Textbook ofIndustrial Microbiology, Second Edition, Sinauer Associates, Inc.,Sunderland, Mass. (1989). All reagents, restriction enzymes andmaterials used for the growth and maintenance of bacterial cells wereobtained from Aldrich Chemicals (Milwaukee, Wis.), BD Diagnostic Systems(Sparks, Md.), Life Technologies (Rockville, Md.), or Sigma ChemicalCompany (St. Louis, Mo.) unless otherwise specified.

Microbial strains were obtained from The American Type CultureCollection (ATCC), Manassas, Va., unless otherwise noted.

The oligonucleotide primers to use in the following Examples are givenin Table 4. All the oligonucleotide primers are synthesized bySigma-Genosys (Woodlands, Tex.).

TABLE 4 Oligonucleotide Cloning, Screening, and  Sequencing Primers SEQID Name Sequence Description NO: N80 CACCATGGACAAACAGTATCCGG budB  11TACGCC forward N81 CGAAGGGCGATAGCTTTACCAAT budB  12 CC reverse N100CACCATGGCTAACTACTTCAATA ilvC   13 CACTGA forward N101CCAGGAGAAGGCCTTGAGTGTTT ilvC   14 TCTCC reverse N102CACCATGCCTAAGTACCGTTCCG ilvD   15 CCACCA forward N103CGCAGCACTGCTCTTAAATATTC ilvD   16 GGC reverse N104CACCATGAACAACTTTAATCTGC yqhD  17 ACACCC forward N105GCTTAGCGGGCGGCTTCGTATAT yqhD  18 ACGGC reverse N110GCATGCCTTAAGAAAGGAGGGG budB  19 GGTCACATGGACAAACAGTATCC forward N111ATGCATTTAATTAATTACAGAATC budB  20 TGACTCAGATGCAGC reverse N112GTCGACGCTAGCAAAGGAGGGA ilvC   21 ATCACCATGGCTAACTACTTCAA forward N113TCTAGATTAACCCGCAACAGCAA ilvC   22 TACGTTTC reverse N114TCTAGAAAAGGAGGAATAAAGTA ilvD   23 TGCCTAAGTACCGTTC forward N115GGATCCTTATTAACCCCCCAGTT ilvD   24 TCGATTTA reverse N116GGATCCAAAGGAGGCTAGACATA kivD   25 TGTATACTGTGGGGGA forward N117GAGCTCTTAGCTTTTATTTTGCTC kivD   26 CGCAAAC reverse N118GAGCTCAAAGGAGGAGCAAGTA yqhD  27 ATGAACAACTTTAATCT forward N119GAATTCACTAGTCCTAGGTTAGC yqhD  28 GGGCGGCTTCGTATATACGG reverse BenNFCAACATTAGCGATTTTCTTTTCTC Npr   29 T forward BenASRCATGAAGCTTACTAGTGGGCTTA Npr   30 AGTTTTGAAAATAATGAAAACT reverse N110.2GAGCTCACTAGTCAATTGTAAGT budB  31 AAGTAAAAGGAGGTGGGTCACAT forwardGGACAAACAGTATCC N111.2 GGATCCGATCGACTTAAGCCTCA budB  32GCTTACAGAATCTGACTCAGATG reverse CAGC N112.2 GAGCTCCTTAAGAAGGAGGTAATilvC   33 CACCATGGCTAACTACTTCAA forward N113.2 GGATCCGATCGAGCTAGCGCGGilvC   34 CCGCTTAACCCGCAACAGCAATA reverse CGTTTC N114.2GAGCTCGCTAGCAAGGAGGTAT ilvD   35 AAAGTATGCCTAAGTACCGTTC forward N115.2GGATCCGATCGATTAATTAACCT ilvD   36 AAGGTTATTAACCCCCCAGTTTC reverse  GATTTA N116.2 GAGCTCTTAATTAAAAGGAGGTT kivD   37 AGACATATGTATACTGTGGGGGAforward N117.2 GGATCCAGATCTCCTAGGACATG kivD   38TTTAGCTTTTATTTTGCTCCGCAA reverse AC N130SeqF1 TGTTCCAACCTGATCACCGsequencing  40 primer N130SeqF2 GGAAAACAGCAAGGCGCT sequencing  41 primerN130SeqF3 CAGCTGAACCAGTTTGCC sequencing  42 primer N130SeqF4AAAATACCAGCGCCTGTCC sequencing  43 primer N130SeqR1 TGAATGGCCACCATGTTGsequencing  44 primer N130SeqR2 GAGGATCTCCGCCGCCTG sequencing  45 primerN130SeqR3 AGGCCGAGCAGGAAGATC sequencing  46 primer N130SeqR4TGATCAGGTTGGAACAGCC sequencing  47 primer N131SeqF1 AAGAACTGATCCCACAGGCsequencing  48 primer N131SeqF2 ATCCTGTGCGGTATGTTGC sequencing  49primer N131SeqF3 ATTGCGATGGTGAAAGCG sequencing  50 primer N131SeqR1ATGGTGTTGGCAATCAGCG sequencing  51 primer N131SeqR2 GTGCTTCGGTGATGGTTTsequencing  52 primer N131SeqR3 TTGAAACCGTGCGAGTAGC sequencing  53primer N132SeqF1 TATTCACTGCCATCTCGCG sequencing  54 primer N132SeqF2CCGTAAGCAGCTGTTCCT sequencing  55 primer N132SeqF3 GCTGGAACAATACGACGTTAsequencing  56 primer N132SeqF4 TGCTCTACCCAACCAGCTTC sequencing  57primer N132SeqR1 ATGGAAAGACCAGAGGTGCC sequencing  58 primer N132SeqR2TGCCTGTGTGGTACGAAT sequencing  59 primer N132SeqR3 TATTACGCGGCAGTGCACTsequencing  60 primer N132SeqR4 GGTGATTTTGTCGCAGTTAGAG sequencing  61primer N133SeqF1 TCGAAATTGTTGGGTCGC sequencing  62 primer N133SeqF2GGTCACGCAGTTCATTTCTAAG sequencing  63 primer N133SeqF3TGTGGCAAGCCGTAGAAA sequencing  64 primer N133SeqF4 AGGATCGCGTGGTGAGTAAsequencing  65 primer N133SeqR1 GTAGCCGTCGTTATTGATGA sequencing  66primer N133SeqR2 GCAGCGAACTAATCAGAGATTC sequencing  67 primer N133SeqR3TGGTCCGATGTATTGGAGG sequencing  68 primer N133SeqR4 TCTGCCATATAGCTCGCGTsequencing  69 primer Scr1 CCTTTCTTTGTGAATCGG sequencing  72 primer Scr2AGAAACAGGGTGTGATCC sequencing  73 primer Scr3 AGTGATCATCACCTGTTGCCsequencing  74 primer Scr4 AGCACGGCGAGAGTCGACGG sequencing  75 primerT-budB AGATAGATGGATCCGGAGGTGG budB 144 (BamHI) GTCACATGGACAAACAGTforward B-kivD CTCTAGAGGATCCAGACTCCTAG kivD  145 (BamHI) GACATG reverseT-groE AGATAGATCTCGAGAGCTATTGT PgroE 147 (XhoI) AACATAATCGGTACGGGGGTGforward B-groEL ATTATGTCAGGATCCACTAGTTT PgroE 148 (SpeI,CCTCCTTTAATTGGGAATTGTTAT reverse BamH1) CCGC T-groELAGCTATTGTAACATAATCGGTAC PgroE 149 GGGGGTG forward T-ilvCB.s.ACATTGATGGATCCCATAACAAG ilvC  150 (BamHI) GGAGAGATTGAAATGGTAAAAG forwardB-iIvCB.s. TAGACAACGGATCCACTAGTTTA ilvC  151 (SpeIATTTTGCGCAACGGAGACCACCG reverse BamHI) C T-BD64 TTACCGTGGACTCACCGAGTGGpBD64 152 (DraIII) GTAACTAGCCTCGCCGGAAAGA forward GCG B-BD64TCACAGTTAAGACACCTGGTGCC pBD64 153 (DraIII) GTTAATGCGCCATGACAGCCATGreverse AT T-Iaclq  ACAGATAGATCACCAGGTGCAAG Iaclq 154 (DraIII)CTAATTCCGGTGGAAACGAGGTC forward ATC B-Iaclq ACAGTACGATACACGGGGTGTCAIaclq 155 (DraIII) CTGCCCGCTTTCCAGTCGGGAAA reverse CC T-groE TCGGATTACGCACCCCGTGAGCT PgroE 156 (DraIII) ATTGTAACATAATCGGTACGGGGforward GTG B-B.s.ilvC CTGCTGATCTCACACCGTGTGTT ilvC  157 (DraIII)AATTTTGCGCAACGGAGACCACC reverse GC T-bdhB TCGATAGCATACACACGGTGGTT bdhB159 (DraIII) AACAAAGGAGGGGTTAAAATGGT forward TGATTTCG B-bdhBATCTACGCACTCGGTGATAAAAC bdhB 160 (rrnBT1 GAAAGGCCCAGTCTTTCGACTGA reverseDraIII) GCCTTTCGTTTTATCTTACACAGA TTTTTTGAATATTTGTAGGAC LDH GACGTCATGACCACCCGCCGATCCC IdhL  161 EcoRV F TTTT forward LDH GATATCCAACACCAGCGACCGACGT IdhL  162 AatIIR ATTAC reverse Cm FATTTAAATCTCGAGTAGAGGATCCCA Cm  163 ACAAACGAAAATTGGATAAAG forward Cm RACGCGTTATTATAAAAGCCAGTCATT Cm  164 AGG reverse P11 F-CCTAGCGCTATAGTTGTTGACAG P11 165 StuI AATGGACATACTATGATATATTGT promoterTGCTATAGCGA forward P11 R- CTAGTCGCTATAGCAACAATATA P11 166 SpeITCATAGTATGTCCATTCTGTCAAC promoter AACTATAGCGCTAGG reverse PIdhL F-AAGCTTGTCGACAAACCAACATT IdhL  167 HindIII ATGACGTGTCTGGGC forwardPIdhL R- GGATCCTCATCCTCTCGTAGTGA IdhL  168 BamHI AAATT reverse F-bdhB-TTCCTAGGAAGGAGGTGGTTAAA bdhB 169 AvrII ATGGTTGATTTCG forward R-bdhB-TTGGATCCTTACACAGATTTTTTG bdhB 170 BamHI AATAT reverse F-ilvCAACTTAAGAAGGAGGTGATTGAA ilvC  171 (B.s.)- ATGGTAAAAGTATATT forward AfIIIR-ilvC AAGCGGCCGCTTAATTTTGCGCA ivIC  172 (B.s.)- ACGGAGACC reverse NotIF- TTAAGCTTGACATACTTGAATGACCT nisA 173 PnisA AGTC promoter (HindIII)forward R-PnisA TTGGATCCAAACTAGTATAATTTATT nisA 174 (SpeI TTGTAGTTCCTTCpromoter BamHI) reverseMethods for Determining Isobutanol Concentration in Culture Media

The concentration of isobutanol in the culture media can be determinedby a number of methods known in the art. For example, a specific highperformance liquid chromatography (HPLC) method utilized a ShodexSH-1011 column with a Shodex SH-G guard column, both purchased fromWaters Corporation (Milford, Mass.), with refractive index (RI)detection. Chromatographic separation was achieved using 0.01 M H₂SO₄ asthe mobile phase with a flow rate of 0.5 mL/min and a column temperatureof 50° C. Isobutanol had a retention time of 46.6 min under theconditions used. Alternatively, gas chromatography (GC) methods areavailable. For example, a specific GC method utilized an HP-INNOWaxcolumn (30 m×0.53 mm id, 1 μm film thickness, Agilent Technologies,Wilmington, Del.), with a flame ionization detector (FID). The carriergas was helium at a flow rate of 4.5 mL/min, measured at 150° C. withconstant head pressure; injector split was 1:25 at 200° C.; oventemperature was 45° C. for 1 min, 45 to 220° C. at 10° C./min, and 220°C. for 5 min; and FID detection was employed at 240° C. with 26 mL/minhelium makeup gas. The retention time of isobutanol was 4.5 min.

The meaning of abbreviations is as follows: “s” means second(s), “min”means minute(s), “h” means hour(s), “psi” means pounds per square inch,“nm” means nanometers, “d” means day(s), “μL” means microliter(s), “mL”means milliliter(s), “L” means liter(s), “mm” means millimeter(s), “nm”means nanometers, “mM” means millimolar, “μM” means micromolar, “M”means molar, “mmol” means millimole(s), “μmol” means micromole(s)”, “g”means gram(s), “μg” means microgram(s) and “ng” means nanogram(s), “PCR”means polymerase chain reaction, “OD” means optical density, “OD₆₀₀”means the optical density measured at a wavelength of 600 nm, “kDa”means kilodaltons, “g” means the gravitation constant, “bp” means basepair(s), “kbp” means kilobase pair(s), “% w/v” means weight/volumepercent, % v/v″ means volume/volume percent, “IPTG” meansisopropyl-8-D-thiogalactopyranoiside, “RBS” means ribosome binding site,“HPLC” means high performance liquid chromatography, and “GC” means gaschromatography. The term “molar selectivity” is the number of moles ofproduct produced per mole of sugar substrate consumed and is reported asa percent.

Example 1 Cloning and Expression of Acetolactate Synthase

The purpose of this Example was to clone the budB gene from Klebsiellapneumoniae and express it in E. coli BL21-AI. The budB gene wasamplified from Klebsiella pneumoniae strain ATCC 25955 genomic DNA usingPCR, resulting in a 1.8 kbp product.

Genomic DNA was prepared using the Gentra Puregene kit (Gentra Systems,Inc., Minneapolis, Minn.; catalog number D-5000A). The budB gene wasamplified from Klebsiella pneumoniae genomic DNA by PCR using primersN80 and N81 (see Table 2), given as SEQ ID NOs:11 and 12, respectively.Other PCR amplification reagents were supplied in manufacturers' kits,for example, Finnzymes Phusion™ High-Fidelity PCR Master Mix (NewEngland Biolabs Inc., Beverly, Mass.; catalog no. F-531) and usedaccording to the manufacturer's protocol. Amplification was carried outin a DNA Thermocycler GeneAmp 9700 (PE Applied Biosystems, Foster city,CA).

For expression studies the Gateway cloning technology (Invitrogen Corp.,Carlsbad, Calif.) was used. The entry vector pENTRSDD-TOPO alloweddirectional cloning and provided a Shine-Dalgarno sequence for the geneof interest. The destination vector pDEST14 used a T7 promoter forexpression of the gene with no tag. The forward primer incorporated fourbases (CACC) immediately adjacent to the translational start codon toallow directional cloning into pENTRSDD-TOPO (Invitrogen) to generatethe plasmid pENTRSDD-TOPObudB. The pENTR construct was transformed intoE. coli Top10 (Invitrogen) cells and plated according to manufacturer'srecommendations. Transformants were grown overnight and plasmid DNA wasprepared using the QIAprep Spin Miniprep kit (Qiagen, Valencia, Calif.;catalog no. 27106) according to manufacturer's recommendations. Cloneswere sequenced to confirm that the genes inserted in the correctorientation and to confirm the sequence. The nucleotide sequence of theopen reading frame (ORF) for this gene and the predicted amino acidsequence of the enzyme are given as SEQ ID NO:1 and SEQ ID NO:2,respectively.

To create an expression clone, the budB gene was transferred to thepDEST 14 vector by recombination to generate pDEST14budB. ThepDEST14budB vector was transformed into E. coli BL21-AI cells(Invitrogen). Transformants were inoculated into Luria Bertani (LB)medium supplemented with 50 μg/mL of ampicillin and grown overnight. Analiquot of the overnight culture was used to inoculate 50 mL of LBsupplemented with 50 μg/mL of ampicillin. The culture was incubated at37° C. with shaking until the OD₆₀₀ reached 0.6-0.8. The culture wassplit into two 25-mL cultures and arabinose was added to one of theflasks to a final concentration of 0.2% w/v. The negative control flaskwas not induced with arabinose. The flasks were incubated for 4 h at 37°C. with shaking. Cells were harvested by centrifugation and the cellpellets were resuspended in 50 mM MOPS, pH 7.0 buffer. The cells weredisrupted either by sonication or by passage through a French PressureCell. The whole cell lysate was centrifuged yielding the supernatant orcell free extract and the pellet or the insoluble fraction. An aliquotof each fraction (whole cell lysate, cell free extract and insolublefraction) was resuspended in SDS (MES) loading buffer (Invitrogen),heated to 85° C. for 10 min and subjected to SDS-PAGE analysis (NuPAGE4-12% Bis-Tris Gel, catalog no. NP0322Box, Invitrogen). A protein of theexpected molecular weight of about 60 kDa, as deduced from the nucleicacid sequence, was present in the induced culture but not in theuninduced control.

Acetolactate synthase activity in the cell free extracts is measuredusing the method described by Bauerle et al. (Biochim. Biophys. Acta92(1):142-149 (1964)).

Example 2 (Prophetic) Cloning and Expression of Acetohydroxy AcidReductoisomerase

The purpose of this prophetic Example is to describe how to clone theilvC gene from E. coli K₁₂ and express it in E. coli BL21-AI. The ilvCgene is amplified from E. coli genomic DNA using PCR.

The ilvC gene is cloned and expressed in the same manner as the budBgene described in Example 1. Genomic DNA from E. coli is prepared usingthe Gentra Puregene kit (Gentra Systems, Inc., Minneapolis, Minn.;catalog number D-5000A). The ilvC gene is amplified by PCR using primersN100 and N101 (see Table 2), given as SEQ ID NOs:13 and 14,respectively, creating a 1.5 kbp product. The forward primerincorporates four bases (CCAC) immediately adjacent to the translationalstart codon to allow directional cloning into pENTR/SD/D-TOPO(Invitrogen) to generate the plasmid pENTRSDD-TOPOilvC. Clones aresequenced to confirm that the genes are inserted in the correctorientation and to confirm the sequence. The nucleotide sequence of theopen reading frame (ORF) for this gene and the predicted amino acidsequence of the enzyme are given as SEQ ID NO:3 and SEQ ID NO:4,respectively.

To create an expression clone, the ilvC gene is transferred to the pDEST14 (Invitrogen) vector by recombination to generate pDEST14ilvC. ThepDEST14ilvC vector is transformed into E. coli BL21-AI cells andexpression from the T7 promoter is induced by addition of arabinose. Aprotein of the expected molecular weight of about 54 kDa, as deducedfrom the nucleic acid sequence, is present in the induced culture, butnot in the uninduced control.

Acetohydroxy acid reductoisomerase activity in the cell free extracts ismeasured using the method described by Arfin and Umbarger (J. Biol.Chem. 244(5):1118-1127 (1969)).

Example 3 (Prophetic) Cloning and Expression of Acetohydroxy AcidDehydratase

The purpose of this prophetic Example is to describe how to clone theilvD gene from E. coli K12 and express it in E. coli BL21-AI. The ilvDgene is amplified from E. coli genomic DNA using PCR.

The ilvD gene is cloned and expressed in the same manner as the budBgene described in Example 1. Genomic DNA from E. coli is prepared usingthe Gentra Puregene kit (Gentra Systems, Inc., Minneapolis, Minn.;catalog number D-5000A). The ilvD gene is amplified by PCR using primersN102 and N103 (see Table 2), given as SEQ ID NOs:15 and 16,respectively, creating a 1.9 kbp product. The forward primerincorporates four bases (CCAC) immediately adjacent to the translationalstart codon to allow directional cloning into pENTR/SD/D-TOPO(Invitrogen) to generate the plasmid pENTRSDD-TOPOiIvD. Clones aresubmitted for sequencing to confirm that the genes are inserted in thecorrect orientation and to confirm the sequence. The nucleotide sequenceof the open reading frame (ORF) for this gene and the predicted aminoacid sequence of the enzyme are given as SEQ ID NO:5 and SEQ ID NO:6,respectively.

To create an expression clone, the ilvD gene is transferred to the pDEST14 (Invitrogen) vector by recombination to generate pDEST14ilvD. ThepDEST14ilvD vector is transformed into E. coli BL21-AI cells andexpression from the T7 promoter is induced by addition of arabinose. Aprotein of the expected molecular weight of about 66 kDa, as deducedfrom the nucleic acid sequence, is present in the induced culture, butnot in the uninduced control.

Acetohydroxy acid dehydratase activity in the cell free extracts ismeasured using the method described by Flint et al. (J. Biol. Chem.268(20):14732-14742 (1993)).

Example 4 (Prophetic) Cloning and Expression of Branched-Chain Keto AcidDecarboxylase

The purpose of this prophetic example is to describe how to clone thekivD gene from Lactococcus lactis and express it in E. coli BL21-AI.

A DNA sequence encoding the branched-chain keto acid decarboxylase(kivD) from L. lactis is obtained from GenScript (Piscataway, N.J.). Thesequence obtained is codon-optimized for expression in both E. coli andB. subtilis and is cloned into pUC57, to form pUC57-kivD. Thecodon-optimized nucleotide sequence of the open reading frame (ORF) forthis gene and the predicted amino acid sequence of the enzyme are givenas SEQ ID NO:7 and SEQ ID NO:8, respectively.

To create an expression clone NdeI and BamHI restriction sites areutilized to clone the 1.7 kbp kivD fragment from pUC57-kivD into vectorpET-3a (Novagen, Madison, Wis.). This creates the expression clonepET-3a-kivD. The pET-3a-kivD vector is transformed into E. coli BL21-AIcells and expression from the T7 promoter is induced by addition ofarabinose. A protein of the expected molecular weight of about 61 kDa,as deduced from the nucleic acid sequence, is present in the inducedculture, but not in the uninduced control.

Branched-chain keto acid decarboxylase activity in the cell freeextracts is measured using the method described by Smit et al. (Appl.Microbiol. Biotechnol. 64:396-402 (2003)).

Example 5 (Prophetic) Cloning and Expression of Branched-Chain AlcoholDehydrogenase

The purpose of this prophetic Example is to describe how to clone theyqhD gene from E. coli K12 and express it in E. coli BL21-AI. The yqhDgene is amplified from E. coli genomic DNA using PCR.

The yqhD gene is cloned and expressed in the same manner as the budBgene described in Example 1. Genomic DNA from E. coli is prepared usingthe Gentra Puregene kit (Gentra Systems, Inc., Minneapolis, Minn.;catalog number D-5000A). The yqhD gene is amplified by PCR using primersN104 and N105 (see Table 2), given as SEQ ID NOs:17 and 18,respectively, creating a 1.2 kbp product. The forward primerincorporates four bases (CCAC) immediately adjacent to the translationalstart codon to allow directional cloning into pENTR/SD/D-TOPO(Invitrogen) to generate the plasmid pENTRSDD-TOPOyqhD. Clones aresubmitted for sequencing to confirm that the genes are inserted in thecorrect orientation and to confirm the sequence. The nucleotide sequenceof the open reading frame (ORF) for this gene and the predicted aminoacid sequence of the enzyme are given as SEQ ID NO 9 and SEQ ID NO:10,respectively.

To create an expression clone, the yqhD gene is transferred to the pDEST14 (Invitrogen) vector by recombination to generate pDEST14yqhD. ThepDEST14ilvD vector is transformed into E. coli BL21-AI cells andexpression from the T7 promoter is induced by addition of arabinose. Aprotein of the expected molecular weight of about 42 kDa, as deducedfrom the nucleic acid sequence, is present in the induced culture, butnot in the uninduced control.

Branched-chain alcohol dehydrogenase activity in the cell free extractsis measured using the method described by Sulzenbacher et al. (J. Mol.Biol. 342(2):489-502 (2004)).

Example 6 (Prophetic) Construction of a Transformation Vector for theGenes in an Isobutanol Biosynthetic Pathway

The purpose of this prophetic Example is to describe how to construct atransformation vector comprising the genes encoding the five steps in anisobutanol biosynthetic pathway. All genes are placed in a single operonunder the control of a single promoter. The individual genes areamplified by PCR with primers that incorporate restriction sites forlater cloning and the forward primers contain an optimized E. coliribosome binding site (AAAGGAGG). PCR products are TOPO cloned into thepCR 4Blunt-TOPO vector and transformed into E. coli Top10 cells(Invitrogen). Plasmid DNA is prepared from the TOPO clones and thesequence of the genes is verified. Restriction enzymes and T4 DNA ligase(New England Biolabs, Beverly, Mass.) are used according tomanufacturer's recommendations. For cloning experiments, restrictionfragments are gel-purified using QIAquick Gel Extraction kit (Qiagen).After confirmation of the sequence, the genes are subcloned into amodified pUC19 vector as a cloning platform. The pUC19 vector ismodified by HindIII/SapI digestion, creating pUC19dHS. The digestremoves the lac promoter adjacent to the MCS (multiple cloning site),preventing transcription of the operons in the vector.

The budB gene is amplified from K. pneumoniae ATCC 25955 genomic DNA byPCR using primer pair N110 and N111 (see Table 2), given as SEQ IDNOs:19 and 20, respectively, creating a 1.8 kbp product. The forwardprimer incorporates SphI and AflII restriction sites and a ribosomebinding site (RBS). The reverse primer incorporates PacI and NsiIrestriction sites. The PCR product is cloned into pCR4 Blunt-TOPOcreating pCR4 Blunt-TOPO-budB. Plasmid DNA is prepared from the TOPOclones and the sequence of the gene is verified. The ilvC gene isamplified from E. coli K12 genomic DNA by PCR using primer pair N112 andN113 (see Table 2) given as SEQ ID NOs:21 and 22, respectively, creatinga 1.5 kbp product. The forward primer incorporates SalI and NheIrestriction sites and a RBS. The reverse primer incorporates a XbaIrestriction site. The PCR product is cloned into pCR4 Blunt-TOPOcreating pCR4 Blunt-TOPO-ilvC. Plasmid DNA is prepared from the TOPOclones and the sequence of the gene is verified.

The ilvD gene is amplified from E. coli K12 genomic DNA by PCR usingprimer pair N114 and N115 (see Table 2) given as SEQ ID NOs:23 and 24,respectively, creating a 1.9 kbp product. The forward primerincorporates a XbaI restriction site and a RBS. The reverse primerincorporates a BamHI restriction site. The PCR product is cloned intopCR4 Blunt-TOPO creating pCR4 Blunt-TOPO-ilvD. Plasmid DNA is preparedfrom the TOPO clones and the sequence of the gene is verified.

The kivD gene is amplified from pUC57-kivD (described in Example 4) byPCR using primer pair N116 and N117 (see Table 2), given as SEQ IDNOs:25 and 26, respectively, creating a 1.7 bp product. The forwardprimer incorporates a BamHI restriction site and a RBS. The reverseprimer incorporates a SacI restriction site. The PCR product is clonedinto pCR4 Blunt-TOPO creating pCR4 Blunt-TOPO-kivD. Plasmid DNA isprepared from the TOPO clones and the sequence of the gene is verified.

The yqhD gene is amplified from E. coli K12 genomic DNA by PCR usingprimer pair N118 and N119 (see Table 2) given as SEQ ID NOs:27 and 28,respectively, creating a 1.2 kbp product. The forward primerincorporates a SacI restriction site. The reverse primer incorporatesSpeI and EcoRI restriction sites. The PCR product is cloned into pCR4Blunt-TOPO creating pCR4 Blunt-TOPO-yqhD. Plasmid DNA is prepared fromthe TOPO clones and the sequence of the gene is verified.

To construct the isobutanol pathway operon, the yqhD gene is excisedfrom pCR4 Blunt-TOPO-yqhD with SacI and EcoRI, releasing a 1.2 kbpfragment. This is ligated with pUC19dHS, which has previously beendigested with SacI and EcoRI. The resulting clone, pUC19dHS-yqhD, isconfirmed by restriction digest. Next, the ilvC gene is excised frompCR4 Blunt-TOPO-ilvC with SalI and XbaI, releasing a 1.5 kbp fragment.This is ligated with pUC19dHS-yqhD, which has previously been digestedwith SalI and XbaI. The resulting clone, pUC19dHS-ilvC-yqhD, isconfirmed by restriction digest. The budB gene is then excised from pCR4Blunt-TOPO-budB with SphI and NsiI, releasing a 1.8 kbp fragment.pUC19dHS-ilvC-yqhD is digested with SphI and PstI and ligated with theSphI/NsiI budB fragment (NsiI and PstI generate compatible ends),forming pUC19dHS-budB-ilvC-yqhD. A 1.9 kbp fragment containing the ilvDgene is excised from pCR4 Blunt-TOPO-ilvD with XbaI and BamHI andligated with pUC19dHS-budB-ilvC-yqhD, which is digested with these sameenzymes, forming pUC19dHS-budB-ilvC-ilvD-yqhD. Finally, kivD is excisedfrom pCR4 Blunt-TOPO-kivD with BamHI and SacI, releasing a 1.7 kbpfragment. This fragment is ligated with pUC19dHS-budB-ilvC-ilvD-yqhD,which has previously been digested with BamHI and SacI, formingpUC19dHS-budB-ilvC-ilvD-kivD-yqhD. The pUC19dHS-budB-ilvC-ilvD-kivD-yqhDvector is digested with AflII and SpeI to release a 8.2 kbp operonfragment that is cloned into pBenAS, an E. coli-B. subtilis shuttlevector. Plasmid pBenAS is created by modification of the pBE93 vector,which is described by Nagarajan, (WO 93/24631, Example 4). To makepBenAS the Bacillus amyloliquefaciens neutral protease promoter (NPR),signal sequence, and the phoA gene are removed with a NcoI/HindIIIdigest of pBE93. The NPR promoter is PCR amplified from pBE93 by primersBenNF and BenASR, given as SEQ ID NOS:29 and 30, respectively. PrimerBenASR incorporates AflII, SpeI, and HindIII sites downstream of thepromoter. The PCR product is digested with NcoI and HindIII and thefragment is cloned into the corresponding sites in the vector creatingpBenAS. The operon fragment is subcloned into the AflII and SpeI sitesin pBenAS creating pBen-budB-ilvC-ilvD-kivD-yqhD.

Example 7 (Prophetic) Expression of the Isobutanol Biosynthetic Pathwayin E. coli

The purpose of this prophetic Example is to describe how to express anisobutanol biosynthetic pathway in E. coli.

The plasmid pBen-budB-ilvC-ilvD-kivD-yqhD, constructed as described inExample 6, is transformed into E. coli NM522 (ATCC No. 47000) to give E.coli strain NM522/pBen-budB-ilvC-ilvD-kivD-yqhD and expression of thegenes in the operon is monitored by SDS-PAGE analysis, enzyme assay andWestern blot analysis. For Western blots, antibodies are raised tosynthetic peptides by Sigma-Genosys (The Woodlands, Tex.).

E. coli strain NM522/pBen-budB-ilvC-ilvD-kivD-yqhD is inoculated into a250 mL shake flask containing 50 mL of medium and shaken at 250 rpm and35° C. The medium is composed of: glucose (5 g/L), MOPS (0.05 M),ammonium sulfate (0.01 M), potassium phosphate, monobasic (0.005 M), S10metal mix (1% (v/v)) yeast extract (0.1% (w/v)), casamino acids (0.1%(w/v)), thiamine (0.1 mg/L), proline (0.05 mg/L), and biotin (0.002mg/L), and is titrated to pH 7.0 with KOH. S10 metal mix contains: MgCl₂(200 mM), CaCl₂ (70 mM), MnCl₂ (5 mM), FeCl₃ (0.1 mM), ZnCl₂ (0.1 mM),thiamine hydrochloride (0.2 mM), CuSO₄ (172 μM), CoCl₂ (253 μM), andNa₂MoO₄ (242 μM). After 18 h, isobutanol is detected by HPLC or GCanalysis, using methods that are well known in the art, for example, asdescribed in the General Methods section above.

Example 8 (Prophetic) Expression of the Isobutanol Biosynthetic Pathwayin Bacillus subtilis

The purpose of this prophetic Example is to describe how to express anisobutanol biosynthetic pathway in Bacillus subtilis. The same approachas described in Example 7 is used.

The plasmid pBen-budB-ilvC-ilvD-kivD-yqhD, constructed as described inExample 6, is used. This plasmid is transformed into Bacillus subtilisBE1010 (J. Bacteriol. 173:2278-2282 (1991)) to give B. subtilis strainBE1010/pBen-budB-ilvC-ilvD-kivD-yqhD and expression of the genes in eachoperon is monitored as described in Example 7.

B. subtilis strain BE1010/pBen-budB-ilvC-ilvD-kivD-yqhD is inoculatedinto a 250 mL shake flask containing 50 mL of medium and shaken at 250rpm and 35° C. for 18 h. The medium is composed of: dextrose (5 g/L),MOPS (0.05 M), glutamic acid (0.02 M), ammonium sulfate (0.01 M),potassium phosphate, monobasic buffer (0.005 M), S10 metal mix (asdescribed in Example 11, 1% (v/v)), yeast extract (0.1% (w/v)), casaminoacids (0.1% (w/v)), tryptophan (50 mg/L), methionine (50 mg/L), andlysine (50 mg/L), and is titrated to pH 7.0 with KOH. After 18 h,isobutanol is detected by HPLC or GC analysis using methods that arewell known in the art, for example, as described in the General Methodssection above.

Example 9 Cloning and Expression of Acetolactate Synthase

To create another acetolactate synthase expression clone, the budB genewas cloned into the vector pTrc99A. The budB gene was first amplifiedfrom pENTRSDD-TOPObudB (described in Example 1) using primers (N110.2and N111.2, given as SEQ ID NOs:31 and 32, respectively) that introducedSacI, SpeI and Mfel sites at the 5′ end and BbvCI, AflII, and BamHIsites at the 3′ end. The resulting 1.75 kbp PCR product was cloned intopCR4-Blunt TOPO (Invitrogen) and the DNA sequence was confirmed (usingN130Seq sequencing primers F1-F4 and R1-R4, given as SEQ ID NOs:40-47,respectively). The budB gene was then excised from this vector usingSacI and BamHI and cloned into pTrc99A (Amann et al. Gene 69(2):301-315(1988)), generating pTrc99A::budB. The pTrc99A::budB vector wastransformed into E. coli TOP10 cells and the transformants wereinoculated into LB medium supplemented with 50 μg/mL of ampicillin andgrown overnight at 37° C.

An aliquot of the overnight culture was used to inoculate 50 mL of LBmedium supplemented with 50 μg/mL of ampicillin. The culture wasincubated at 37° C. with shaking until the OD₆₀₀ reached 0.6 to 0.8.Expression of budB from the Trc promoter was then induced by theaddition of 0.4 mM IPTG. Negative control flasks were also prepared thatwere not induced with IPTG. The flasks were incubated for 4 h at 37° C.with shaking. Cell-free extracts were prepared as described in Example1.

Acetolactate synthase activity in the cell free extracts was measured asdescribed in Example 1. Three hours after induction with IPTG, anacetolactate synthase activity of 8 units/mg was detected. The controlstrain carrying only the pTrc99A plasmid exhibited 0.03 units/mg ofacetolactate synthase activity.

Example 10 Cloning and Expression of Acetohydroxy Acid Reductoisomerase

The purpose of this Example was to clone the ilvC gene from E. coli K12and express it in E. coli TOP10. The ilvC gene was amplified from E.coli K12 strain FM5 (ATCC 53911) genomic DNA using PCR.

The ilvC gene was cloned and expressed in a similar manner as describedfor the cloning and expression of ilvC in Example 2 above. PCR was usedto amplify ilvC from the E. coli FM5 genome using primers N112.2 andN113.2 (SEQ ID NOs:33 and 34, respectively). The primers created SacIand AMU sites and an optimal RBS at the 5′ end and NotI, NheI and BamHIsites at the 3′ end of ilvC. The 1.5 kbp PCR product was cloned intopCR4Blunt TOPO according to the manufacturer's protocol (Invitrogen)generating pCR4Blunt TOPO::ilvC. The sequence of the PCR product wasconfirmed using sequencing primers (N131SeqF1-F3, and N131SeqR1-R3,given as SEQ ID NOs:48-53, respectively). To create an expression clone,the ilvC gene was excised from pCR4Blunt TOPO::ilvC using SacI and BamHIand cloned into pTrc99A. The pTrc99A::ilvC vector was transformed intoE. coli TOP10 cells and expression from the Trc promoter was induced byaddition of IPTG, as described in Example 9. Cell-free extracts wereprepared as described in Example 1.

Acetohydroxy acid reductoisomerase activity in the cell free extractswas measured as described in Example 2. Three hours after induction withIPTG, an acetohydroxy acid reductoisomerase activity of 0.026 units/mgwas detected. The control strain carrying only the pTrc99A plasmidexhibited less than 0.001 units/mg of acetohydroxy acid reductoisomeraseactivity.

Example 11 Cloning and Expression of Acetohydroxy Acid Dehydratase

The purpose of this Example was to clone the ilvD gene from E. coli K12and express it in E. coli Top10. The ilvD gene was amplified from E.coli K12 strain FM5 (ATCC 53911) genomic DNA using PCR.

The ilvD gene was cloned and expressed in a similar manner as the ilvCgene described in Example 10. PCR was used to amplify ilvD from the E.coli FM5 genome using primers N114.2 and N115.2 (SEQ ID NOs:35 and 36,respectively). The primers created SacI and NheI sites and an optimalRBS at the 5′ end and Bsu36I, PacI and BamHI sites at the 3′ end ofilvD. The 1.9 kbp PCR product was cloned into pCR4Blunt TOPO accordingto the manufacturer's protocol (Invitrogen) generating pCR4BluntTOPO::ilvD. The sequence of the PCR product was confirmed (sequencingprimers N132SeqF1-F4 and N132SeqR1-R4, given as SEQ ID NOs:54-61,respectively). To create an expression clone, the ilvD gene was excisedfrom plasmid pCR4Blunt TOPO::ilvD using SacI and BamHI, and cloned intopTrc99A. The pTrc99A::ilvD vector was transformed into E. coli TOP10cells and expression from the Trc promoter was induced by addition ofIPTG, as described in Example 9. Cell-free extracts were prepared asdescribed in Example 1.

Acetohydroxy acid dehydratase activity in the cell free extracts wasmeasured as described in Example 3. Three hours after induction withIPTG, an acetohydroxy acid dehydratase activity of 46 units/mg wasmeasured. The control strain carrying only the pTrc99A plasmid exhibitedno detectable acetohydroxy acid dehydratase activity.

Example 12 Cloning and Expression of Branched-Chain Keto AcidDecarboxylase

The purpose of this Example was to clone the kivD gene from Lactococcuslactis and express it in E. coli TOP10.

The kivD gene was cloned and expressed in a similar manner as thatdescribed for ilvC in Example 10 above. PCR was used to amplify kivDfrom the plasmid pUC57-kivD (see Example 4, above) using primers N116.2and N117.2 (SEQ ID NOs:37 and 38, respectively). The primers createdSacI and PacI sites and an optimal RBS at the 5′ end and PciI, AvrII,BglII and BamHI sites at the 3′ end of kivD. The 1.7 kbp PCR product wascloned into pCR4Blunt TOPO according to the manufacturer's protocol(Invitrogen) generating pCR4Blunt TOPO::kivD. The sequence of the PCRproduct was confirmed using primers N133SeqF1-F4 and N133SeqR1-R4 (givenas SEQ ID NOs:62-69, respectively). To create an expression clone, thekivD gene was excised from plasmid pCR4Blunt TOPO::kivD using SacI andBamHI, and cloned into pTrc99A. The pTrc99A::kivD vector was transformedinto E. coli TOP10 cells and expression from the Trc promoter wasinduced by addition of IPTG, as described in Example 9. Cell-freeextracts were prepared as described in Example 1.

Branched-chain keto acid decarboxylase activity in the cell freeextracts was measured as described in Example 4, except that Purpald®reagent (Aldrich, Catalog No. 162892) was used to detect and quantifythe aldehyde reaction products. Three hours after induction with IPTG, abranched-chain keto acid decarboxylase activity of greater than 3.7units/mg was detected. The control strain carrying only the pTrc99Aplasmid exhibited no detectable branched-chain keto acid decarboxylaseactivity.

Example 13 Expression of Branched-Chain Alcohol Dehydrogenase

E. coli contains a native gene (yqhD) that was identified as a1,3-propanediol dehydrogenase (U.S. Pat. No. 6,514,733). The YqhDprotein has 40% identity to AdhB (encoded by adhB) from Clostridium, aputative NADH-dependent butanol dehydrogenase. The yqhD gene was placedunder the constitutive expression of a variant of the glucose isomerasepromoter 1.6GI (SEQ ID NO. 70) in E. coli strain MG1655 1.6yqhD::Cm (WO2004/033646) using λ Red technology (Datsenko and Wanner, Proc. Natl.Acad. Sci. U.S.A. 97:6640 (2000)). MG1655 1.6yqhD::Cm contains aFRT-CmR-FRT cassette so that the antibiotic marker can be removed.Similarly, the native promoter was replaced by the 1.5GI promoter (WO2003/089621) (SEQ ID NO. 71), creating strain

MG1655 1.5GI-yqhD::Cm, thus, replacing the 1.6GI promoter of MG16551.6yqhD::Cm with the 1.5GI promoter.

Strain MG1655 1.5GI-yqhD::Cm was grown in LB medium to mid-log phase andcell free extracts were prepared as described in Example 1. This strainwas found to have NADPH-dependent isobutyraldehyde reductase activitywhen the cell extracts were assayed by following the decrease inabsorbance at 340 nm at pH 7.5 and 35° C.

To generate a second expression strain containing 1.5GI yqhD::Cm, a P1lysate was prepared from MG1655 1.5GI yqhD::Cm and the cassette wastransferred to BL21 (DE3) (Invitrogen) by transduction, creating BL21(DE3) 1.5GI-yqhD::Cm.

Example 14 Construction of a Transformation Vector for the First FourGenes in an Isobutanol Biosynthetic Pathway

The purpose of this Example was to construct a transformation vectorcomprising the first four genes (i.e., budB, ilvC, ilvD and kivD) in anisobutanol biosynthetic pathway.

To construct the transformation vector, first, the ilvC gene wasobtained from pTrc99A::ilvC (described in Example 10) by digestion withAflII and BamHI and cloned into pTrc99A::budB (described in Example 9),which was digested with AflII and BamHI to produce plasmidpTrc99A::budB-ilvC. Next, the ilvD and kivD genes were obtained frompTrc99A::ilvD (described in Example 11) and pTrc99A::kivD (described inExample 12), respectively, by digestion with NheI and PacI (ilvD) andPacI and BamHI (kivD). These genes were introduced intopTrc99A::budB-ilvC, which was first digested with NheI and BamHI, bythree-way ligation. The presence of all four genes in the final plasmid,pTrc99A::budB-ilvC-ilvD-kivD, was confirmed by PCR screening andrestriction digestion.

Example 15 Expression of an Isobutanol Biosynthetic Pathway in E. coliGrown on Glucose

To create E. coli isobutanol production strains,pTrc99A::budB-ilvC-ilvD-kivD (described in Example 14) was transformedinto E. coli MG1655 1.5GI yqhD::Cm and E. coli BL21 (DE3) 1.5GI yqhD::Cm(described in Example 13). Transformants were initially grown in LBmedium containing 50 μg/mL kanamycin and 100 μg/mL carbenicillin. Thecells from these cultures were used to inoculate shake flasks(approximately 175 mL total volume) containing 50 or 170 mL ofTM3a/glucose medium (with appropriate antibiotics) to represent high andlow oxygen conditions, respectively. TM3a/glucose medium contained (perliter): glucose (10 g), KH₂PO₄ (13.6 g), citric acid monohydrate (2.0g), (NH₄)₂SO₄ (3.0 g), MgSO₄.7H₂O (2.0 g), CaCl₂.2H₂O (0.2 g), ferricammonium citrate (0.33 g), thiamine.HCl (1.0 mg), yeast extract (0.50g), and 10 mL of trace elements solution. The pH was adjusted to 6.8with NH₄OH. The trace elements solution contained: citric acid.H₂O (4.0g/L), MnSO₄.H₂O (3.0 g/L), NaCl (1.0 g/L), FeSO₄.7H₂O (0.10 g/L),CoCl₂.6H₂O (0.10 g/L), ZnSO₄.7H₂O (0.10 g/L), CuSO₄.5H₂O (0.010 g/L),H₃BO₃ (0.010 g/L), and Na₂MoO₄. 2H₂O (0.010 g/L).

The flasks were inoculated at a starting OD₆₀₀ of ≦0.01 units andincubated at 34° C. with shaking at 300 rpm. The flasks containing 50 mLof medium were closed with 0.2 μm filter caps; the flasks containing 150mL of medium were closed with sealed caps. IPTG was added to a finalconcentration of 0.04 mM when the cells reached an OD₆₀₀ of ≧0.4 units.Approximately 18 h after induction, an aliquot of the broth was analyzedby HPLC (Shodex Sugar SH1011 column (Showa Denko America, Inc. NY) withrefractive index (RI) detection) and GC (Varian CP-WAX 58(FFAP) CB, 0.25mm×0.2 μm×25 m (Varian, Inc., Palo Alto, Calif.) with flame ionizationdetection (FID)) for isobutanol content, as described in the GeneralMethods section. No isobutanol was detected in control strains carryingonly the pTrc99A vector (results not shown). Molar selectivities andtiters of isobutanol produced by strains carryingpTrc99A::budB-ilvC-ilvD-kivD are shown in Table 5. Significantly highertiters of isobutanol were obtained in the cultures grown under lowoxygen conditions.

TABLE 5 Production of Isobutanol by E. coli Strains Grown on GlucoseMolar O₂ Isobutanol Selectivity Strain Conditions mM* (%) MG1655 1.5GIyqhD/ High 0.4 4.2 pTrc99A::budB-ilvC-ilvD-kivD MG1655 1.5GI yqhD/ Low9.9 39 pTrc99A::budB-ilvC-ilvD-kivD BL21 (DE3) 1.5GI yqhD/ High 0.3 3.9pTrc99A::budB-ilvC-ilvD-kivD BL21 (DE3) 1.5GI yqhD/ Low 1.2 12pTrc99A::budB-ilvC-ilvD-kivD *Determined by HPLC.

Example 16 Expression of an Isobutanol Biosynthetic Pathway in E. coliGrown on Sucrose

Since the strains described in Example 15 were not capable of growth onsucrose, an additional plasmid was constructed to allow utilization ofsucrose for isobutanol production. A sucrose utilization gene clustercscBKA, given as SEQ ID NO:39, was isolated from genomic DNA of asucrose-utilizing E. coli strain derived from ATCC strain 13281. Thesucrose utilization genes (cscA, cscK, and cscB) encode a sucrosehydrolase (CscA), given as SEQ ID NO:139, D-fructokinase (CscK), givenas SEQ ID NO:140, and sucrose permease (CscB), given as SEQ ID NO:141.The sucrose-specific repressor gene cscR was not included so that thethree genes cscBKA were expressed constitutively from their nativepromoters in E. coli.

Genomic DNA from the sucrose-utilizing E. coli strain was digested tocompletion with BamHI and EcoRI. Fragments having an average size ofabout 4 kbp were isolated from an agarose gel and were ligated toplasmid pLitmus28 (New England Biolabs), digested with BamHI and EcoRIand transformed into ultracompetent E. coli TOP10F′ cells (Invitrogen).The transformants were streaked onto MacConkey agar plates containing 1%sucrose and ampicillin (100 μg/mL) and screened for the appearance ofpurple colonies. Plasmid DNA was isolated from the purple transformants,and sequenced with M13 Forward and Reverse primers (Invitrogen), andScr1-4 (given as SEQ ID NOs:72-75, respectively). The plasmid containingcscB, cscK, and cscA (cscBKA) genes was designated pScr1.

To create a sucrose utilization plasmid that was compatible with theisobutanol pathway plasmid (Example 14), the operon from pScr1 wassubcloned into pBHR1 (MoBiTec, Goettingen, Germany). The cscBKA geneswere isolated by digestion of pScr1 with XhoI (followed by incubationwith Klenow enzyme to generate blunt ends) and then by digestion withAgeI. The resulting 4.2 kbp fragment was ligated into pBHR1 that hadbeen digested with Nael and AgeI, resulting in the 9.3 kbp plasmidpBHR1::cscBKA.

The sucrose plasmid pBHR1::cscBKA was transformed into E. coli BL21(DE3) 1.5 yqhD/pTrc99A::budB-ilvC-ilvD-kivD and E. coli MG16551.5yqhD/pTrc99A::budB-ilvC-ilvD-kivD (described in Example 15) byelectroporation. Transformants were first selected on LB mediumcontaining 100 μg/mL ampicillin and 50 μg/mL kanamycin and then screenedon MacConkey sucrose (1%) plates to confirm functional expression of thesucrose operon. For production of isobutanol, strains were grown in TM3aminimal defined medium (described in Example 15) containing 1% sucroseinstead of glucose, and the culture medium was analyzed for the amountof isobutanol produced, as described in Example 15, except that sampleswere taken 14 h after induction. Again, no isobutanol was detected incontrol strains carrying only the pTrc99A vector (results not shown).Molar selectivities and titers of isobutanol produced by MG1655 1.5yqhDcarrying pTrc99A::budB-ilvC-ilvD-kivD are shown in Table 6. Similarresults were obtained with the analogous BL21 (DE3) strain.

TABLE 6 Production of Isobutanol by E. coli strain MG16551.5yqhD/pTrc99A:: budB-ilvC-ilvD-kivD/pBHR1::cscBKA Grown on Sucrose O₂Isobutanol, Molar Conditions IPTG, mM mM* Selectivity, % High 0.04 0.172 High 0.4 1.59 21 Low 0.04 4.03 26 Low 0.4 3.95 29 *Determined by HPLC.

Example 17 Expression of Isobutanol Pathway Genes in SaccharomycesCerevisiae

To express isobutanol pathway genes in Saccharomyces cerevisiae, anumber of E. coli-yeast shuttle vectors were constructed. A PCR approach(Yu, et al. Fungal Genet. Biol. 41:973-981 (2004)) was used to fusegenes with yeast promoters and terminators. Specifically, the GPDpromoter (SEQ ID NO:76) and CYC1 terminator (SEQ ID NO:77) were fused tothe alsS gene from Bacillus subtilis (SEQ ID NO:78), the FBA promoter(SEQ ID NO:79) and CYC1 terminator were fused to the ILV5 gene from S.cerevisiae (SEQ ID NO:80), the ADH1 promoter (SEQ ID NO:81) and ADH1terminator (SEQ ID NO:82) were fused to the ILV3 gene from S. cerevisiae(SEQ ID NO:83), and the GPM promoter (SEQ ID NO:84) and ADH1 terminatorwere fused to the kivD gene from Lactococcus lactis (SEQ ID NO:7). Theprimers, given in Table 7, were designed to include restriction sitesfor cloning promoter/gene/terminator products into E. coli-yeast shuttlevectors from the pRS400 series (Christianson et al. Gene 110:119-122(1992)) and for exchanging promoters between constructs. Primers for the5′ ends of ILV5 and ILV3 (N138 and N155, respectively, given as SEQ IDNOs: 95 and 107, respectively) generated new start codons to eliminatemitochondrial targeting of these enzymes.

All fused PCR products were first cloned into pCR4-Blunt by TOPO cloningreaction (Invitrogen) and the sequences were confirmed (using M13forward and reverse primers (Invitrogen) and the sequencing primersprovided in Table 7. Two additional promoters (CUP1 and GAL1) werecloned by TOPO reaction into pCR4-Blunt and confirmed by sequencing;primer sequences are indicated in Table 7. The plasmids that wereconstructed are described in Table 8. The plasmids were transformed intoeither Saccharomyces cerevisiae BY4743 (ATCC 201390) or YJR148w (ATCC4036939) to assess enzyme specific activities using the enzyme assaysdescribed in Examples 1-4 and Examples 9-12. For the determination ofenzyme activities, cultures were grown to an OD₆₀₀ of 1.0 in syntheticcomplete medium (Methods in Yeast Genetics, 2005, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., pp. 201-202) lacking anymetabolite(s) necessary for selection of the expression plasmid(s),harvested by centrifugation (2600×g for 8 min at 4° C.), washed withbuffer, centrifuged again, and frozen at −80° C. The cells were thawed,resuspended in 20 mM Tris-HCl, pH 8.0 to a final volume of 2 mL, andthen disrupted using a bead beater with 1.2 g of glass beads (0.5 mmsize). Each sample was processed on high speed for 3 minutes total (withincubation on ice after each minute of beating). Extracts were clearedof cell debris by centrifugation (20,000×g for 10 min at 4° C.).

TABLE 7 Primer Sequences for Cloning and Sequencing ofS. cerevisiae Expression Vectors SEQ ID Name Sequence Description NO:N98SeqF1 CGTGTTAGTCACATCAGGA B. subtilis alsS  85 C sequencing primerN98SeqF2 GGCCATAGCAAAAATCCAA B. subtilis alsS  86 ACAGCsequencing primer N98SeqF3 CCACGATCAATCATATCGA B. subtilis alsS  87ACACG sequencing primer N98SeqF4 GGTTTCTGTCTCTGGTGAC B. subtilis alsS 88 G sequencing primer N99SeqR1 GTCTGGTGATTCTACGCGC B. subtilis alsS 89 AAG sequencing primer N99SeqR2 CATCGACTGCATTACGCAA B. subtilis alsS 90 CTC sequencing primer N99SeqR3 CGATCGTCAGAACAACATC B. subtilis alsS 91 TGC sequencing primer N99SeqR4 CCTTCAGTGTTCGCTGTCA B. subtilis alsS 92 G sequencing primer N136 CCGCGGATAGATCTGAAAT FBA promoter  93GAATAACAATACTGACA forward primer with SaclI/BglII sites N137TACCACCGAAGTTGATTTG FBA promoter  94 CTTCAACATCCTCAGCTCT reverse primerAGATTTGAATATGTATTACT with BbvCI site TGGTTAT and ILV5- annealing regionN138 ATGTTGAAGCAAATCAACT ILV5 forward  95 TCGGTGGTA primer (createsalternate start codon) N139 TTATTGGTTTTCTGGTCTCA ILV5 reverse  96 ACprimer N140 AAGTTGAGACCAGAAAACC CYC terminator  97 AATAATTAATTAATCATGTAforward primer ATTAGTTATGTCACGCTT with PacI  site and ILV5-annealingregion N141 GCGGCCGCCCGCAAATTA CYC terminator  98 AAGCCTTCGAGCreverse primer with NotI site N142 GGATCCGCATGCTTGCATT GPM promoter  99TAGTCGTGC forward primer with BamHI site N143 CAGGTAATCCCCCACAGTAGPM promoter 100 TACATCCTCAGCTATTGTA reverse primer ATATGTGTGTTTGTTTGGwith BbvCI site and kivD- annealing region N144 ATGTATACTGTGGGGGATTkivD forward 101 ACC primer N145 TTAGCTTTTATTTTGCTCCG kivD reverse 102CA primer N146 TTTGCGGAGCAAAATAAAA ADH terminator 103GCTAATTAATTAAGAGTAA forward primer GCGAATTTCTTATGATTTA with PacI site and kivD-annealing region N147 ACTAGTACCACAGGTGTTG ADH terminator104 TCCTCTGAG reverse primer with SpeI site N151 CTAGAGAGCTTTCGTTTTCalsS reverse 105 ATG primer N152 CTCATGAAAACGAAAGCTC CYC terminator 106TCTAGTTAATTAATCATGTA forward primer ATTAGTTATGTCACGCTT with PacI site and alsS-annealing region N155 ATGGCAAAGAAGCTCAACA ILV3 forward 107AGTACT primer (alternate start codon) N156 TCAAGCATCTAAAACACAAILV3 reverse 108 CCG primer N157 AACGGTTGTGTTTTAGATG ADH terminator 109CTTGATTAATTAAGAGTAA forward primer GCGAATTTCTTATGATTTA with PacI site and ILV3-annealing region N158 GGATCCTTTTCTGGCAACC ADH promoter 110AAACCCATA forward primer with BamHI site N159 CGAGTACTTGTTGAGCTTCADH promoter 111 TTTGCCATCCTCAGCGAGA reverse primer TAGTTGATTGTATGCTTGwith BbvCI site and ILV3- annealing region N160SeqF1 GAAAACGTGGCATCCTCTCFBA::ILV5::CYC 112 sequencing primer N160SeqF2 GCTGACTGGCCAAGAGAAFBA::ILV5::CYC 113 A sequencing primer N160SeqF3 TGTACTTCTCCCACGGTTTFBA::ILV5::CYC 114 C sequencing primer N160SeqF4 AGCTACCCAATCTCTATACFBA::ILV5::CYC 115 CCA sequencing primer N160SeqF5 CCTGAAGTCTAGGTCCCTAFBA::ILV5::CYC 116 TTT sequencing primer N160SeqR1 GCGTGAATGTAAGCGTGAFBA::ILV5::CYC 117 C sequencing primer N160SeqR2 CGTCGTATTGAGCCAAGAAFBA::ILV5::CYC 118 C sequencing primer N160SeqR3 GCATCGGACAACAAGTTCAFBA::ILV5::CYC 119 T sequencing primer N160SeqR4 TCGTTCTTGAAGTAGTCCAFBA::ILV5::CYC 120 ACA sequencing primer N160SeqR5 TGAGCCCGAAAGAGAGGAFBA::ILV5::CYC 121 T sequencing primer N161SeqF1 ACGGTATACGGCCTTCCTTADH::ILV3::ADH 122 sequencing primer Nl6lSeqF2 GGGTTTGAAAGCTATGCAGADH::ILV3::ADH 123 T sequencing primer N161SeqF3 GGTGGTATGTATACTGCCAADH::ILV3::ADH 124 ACA sequencing primer N161SeqF4 GGTGGTACCCAATCTGTGAADH::ILV3::ADH 125 TTA sequencing primer N16lSeqF5 CGGTTTGGGTAAAGATGTTADH::ILV3::ADH 126 G sequencing primer N161SeqF6 AAACGAAAATTCTTATTCTTADH::ILV3::ADH 127 GA sequencing primer N161SeqR1 TCGTTTTAAAACCTAAGAGADH::ILV3::ADH 128 TCA sequencing primer N161SeqR2 CCAAACCGTAACCCATCAGADH::ILV3::ADH 129 sequencing primer N161SeqR3 CACAGATTGGGTACCACCAADH::ILV3::ADH 130 sequencing primer N161SeqR4 ACCACAAGAACCAGGACCTADH::ILV3::ADH 131 G sequencing primer N161SeqR5 CATAGCTTTCAAACCCGCTADH::ILV3::ADH 132 sequencing primer N161SeqR6 CGTATACCGTTGCTCATTAADH::ILV3::ADH 133 GAG sequencing primer N162 ATGTTGACAAAAGCAACAAa/sS forward 134 AAGA primer N189 ATCCGCGGATAGATCTAGT GPD forward 135TCGAGTTTATCATTATCAA primer with SaclI/BglII sites N190.1TTCTTTTGTTGCTTTTGTCA GPD promoter 136 ACATCCTCAGCGTTTATGT reverse primerGTGTTTATTCGAAA with BbvCI site and alsS- annealing region N176ATCCGCGGATAGATCTATT GAL1 promoter 137 AGAAGCCGCCGAGCGGGC forward primerG with SaclI/BglII sites N177 ATCCTCAGCTTTTCTCCTT GAL1 promoter 138GACGTTAAAGTA reverse with  BbvCI site N191 ATCCGCGGATAGATCTCCCCUP1 promoter 175 ATTACCGACATTTGGGCGC forward primer with SaclI/BglIIsites N192 ATCCTCAGCGATGATTGAT CUP1 promoter 176 TGATTGATTGTAreverse with  BbvCI site

TABLE 8 E. coli-Yeast Shuttle Vectors Carrying Isobutanol Pathway GenesPlasmid Name Construction pRS426 [ATCC No. 77107], — URA3 selectionpRS426::GPD::alsS::CYC GPD::alsS::CYC PCR product digested withSacII/NotI cloned into pRS426 digested with same pRS426::FBA::ILV5::CYCFBA::ILV5::CYC PCR product digested with SacII/NotI cloned into pRS426digested with same pRS425 [ATCC No. 77106], — LEU2 selectionpRS425::ADH::ILV3::ADH ADH::ILV3::ADH PCR product digested withBamHI/SpeI cloned into pRS425 digested with same pRS425::GPM::kivD::ADHGPM::kivD::ADH PCR product digested with BamHI/SpeI cloned into pRS425digested with same pRS426::CUP1::alsS 7.7 kbp SacII/BbvCI fragment frompRS426::GPD::alsS::CYC ligated with SacII/BbvCI CUP1 fragmentpRS426::GAL1::ILV5 7 kbp SacII/BbvCI fragment frompRS426::FBA::ILV5::CYC ligated with SacII/BbvCI GAL1 fragmentpRS425::FBA::ILV3 8.9 kbp BamHI/BbvCI fragment frompRS425::ADH::ILV3::ADH ligated with 0.65 kbp BglII/BbvCI FBA fragmentfrom pRS426::FBA::ILV5::CYC pRS425::CUP1-alsS + FBA-ILV3 2.4 kbpSacII/NotI fragment from pRS426::CUP1::alsS cloned intopRS425::FBA::ILV3 cut with SacII/NotI pRS426::FBA-ILV5 + GPM-kivD 2.7kbp BamHI/SpeI fragment from pRS425::GPM::kivD::ADH cloned intopRS426::FBA::ILV5::CYC cut with BamHI/SpeI pRS426::GAL1-FBA + GPM-kivD8.5 kbp SacII/NotI fragment from pRS426:: FBA- ILV5 + GPM-kivD ligatedwith 1.8 kbp SacII/NotI fragment from pRS426::GAL1::ILV5 pRS423 [ATCCNo. 77104], — HIS3 selection pRS423::CUP1-alsS + FBA-ILV3 5.2 kbpSacI/SalI fragment from pRS425::CUP1- alsS + FBA-ILV3 ligated intopRS423 cut with SacI/SalI pHR81 [ATCC No. 87541], — URA3 and leu2-dselection pHR81::FBA-ILV5 + GPM- 4.7 kbp SacI/BamHI fragment frompRS426::FBA- kivD ILV5 + GPM-kivD ligated into pHR81 cut with SacI/BamHI

Example 18 Production of Isobutanol by Recombinant SaccharomycesCerevisiae

Plasmids pRS423::CUP1-alsS+FBA-ILV3 and pHR81::FBA-ILV5+GPM-kivD(described in Example 17) were transformed into Saccharomyces cerevisiaeYJR148w to produce strainYJR148w/pRS423::CUP1-alsS+FBA-ILV3/pHR81::FBA-ILV5+GPM-kivD. A controlstrain was prepared by transforming vectors pRS423 and pHR81 (describedin Example 17) into Saccharomyces cerevisiae YJR148w (strainYJR148w/pRS423/pHR81). Strains were maintained on standard S. cerevisiaesynthetic complete medium (Methods in Yeast Genetics, 2005, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., pp. 201-202)containing either 2% glucose or sucrose but lacking uracil and histidineto ensure maintenance of plasmids.

For isobutanol production, cells were transferred to synthetic completemedium lacking uracil, histidine and leucine. Removal of leucine fromthe medium was intended to trigger an increase in copy number of thepHR81-based plasmid due to poor transcription of the leu2-d allele(Erhart and Hollenberg, J. Bacteriol. 156:625-635 (1983)). Aerobiccultures were grown in 175 mL capacity flasks containing 50 mL of mediumin an Innova4000 incubator (New Brunswick Scientific, Edison, N.J.) at30° C. and 200 rpm. Low oxygen cultures were prepared by adding 45 mL ofmedium to 60 mL serum vials that were sealed with crimped caps afterinoculation and kept at 30° C. Sterile syringes were used for samplingand addition of inducer, as needed. Approximately 24 h afterinoculation, the inducer CuSO₄ was added to a final concentration of0.03 mM. Control cultures for each strain without CuSO₄ addition werealso prepared. Culture supernatants were analyzed 18 or 19 h and 35 hafter CuSO₄ addition by both GC and HPLC for isobutanol content, asdescribed above in Example 15. The results for S. cerevisiaeYJR148w/pRS423::CUP1-alsS+FBA-ILV3/pHR81::FBA-ILV5+GPM-kivD grown onglucose are presented in Table 9. For the results given in Table 9, thesamples from the aerobic cultures were taken at 35 h and the samplesfrom the low oxygen cultures were taken at 19 h and measured by HPLC.

The results for S. cerevisiaeYJR148w/pRS423::CUP1-alsS+FBA-ILV3/pHR81::FBA-ILV5+GPM-kivD grown onsucrose are presented in Table 10. The results in this table wereobtained with samples taken at 18 h and measured by HPLC.

TABLE 9 Production of Isobutanol by S. cerevisiae YJR148w/pRS423::CUP1-alsS + FBA-ILV3/pHR81::FBA-ILV5 + GPM-kivD Grown on Glucose MolarIsobutanol, Selectivity Strain O₂ level mM % YJR148w/pRS423/pHR81(control) Aerobic 0.12 0.04 YJR148w/pRS423/pHR81 (control) Aerobic 0.110.04 YJR148w/pRS423::CUP1-alsS + Aerobic 0.97 0.34IFBA-LV3/pHR81::FBA-ILV5 + GPM-kivD a YJR148w/pRS423::CUP1-alsS +Aerobic 0.93 0.33 FBA-ILV3/pHR81::FBA-ILV5 + GPM-kivD bYJR148w/pRS423::CUP1-alsS + Aerobic 0.85 0.30 FBA-ILV3/pHR81::FBA-ILV5 +GPM-kivD c YJR148w/pRS423/pHR81 (control) Low 0.11 0.1YJR148w/pRS423/pHR81 (control) Low 0.08 0.1 YJR148w/pRS423::CUP1-alsS +Low 0.28 0.5 FBA-ILV3/pHR81::FBA-ILV5 + GPM-kivD aYJR148w/pRS423::CUP1-alsS + Low 0.20 0.3 FBA-ILV3/pHR81::FBA-ILV5 +GPM-kivD b YJR148w/pRS423::CUP1-alsS + Low 0.33 0.6FBA-ILV3/pHR81::FBA-ILV5 + GPM-kivD c

TABLE 10 Production of Isobutanol by S. cerevisiae YJR148w/pRS423::CUP1-alsS + FBA-ILV3/pHR81::FBA-ILV5 + GPM-kivD Grown on Sucrose Molar O₂Isobutanol Selectivity, Strain Level mM % YJR148w/pRS423/pHR81 (control)Aerobic 0.32 0.6 YJR148w/pRS423/pHR81 (control) Aerobic 0.17 0.3YJR148w/pRS423::CUP1-alsS + Aerobic 0.68 1.7 IFBA-LV3/pHR81::FBA-ILV5 +GPM-kivD a YJR148w/pRS423::CUP1-alsS + Aerobic 0.54 1.2FBA-ILV3/pHR81::FBA-ILV5 + GPM-kivD b YJR148w/pRS423::CUP1-alsS +Aerobic 0.92 2.0 IFBA-LV3/pHR81::FBA-ILV5 + GPM-kivD cYJR148w/pRS423/pHR81 (control) Low 0.18 0.3 YJR148w/pRS423/pHR81(control) Low 0.15 0.3 YJR148w/pRS423::CUP1-alsS + Low 0.27 1.2FBA-ILV3/pHR81::FBA-ILV5 + GPM-kivD a YJR148w/pRS423::CUP1-alsS + Low0.30 1.1 FBA-ILV3/pHR81::FBA-ILV5 + GPM-kivD bYJR148w/pRS423::CUP1-alsS + Low 0.21 0.8 FBA-ILV3/pHR81::FBA-ILV5 +GPM-kivD c Strain suffixes “a”, “b”, and “c” indicate separate isolates.

The results indicate that, when grown on glucose or sucrose under bothaerobic and low oxygen conditions, strainYJR148w/pRS423::CUP1-alsS+FBA-ILV3/pHR81::FBA-ILV5+GPM-kivD producedconsistently higher levels of isobutanol than the control strain.

Example 19 Production of Isobutanol by Recombinant SaccharomycesCerevisiae

Plasmids pRS425::CUP1-alsS+FBA-ILV3 and pRS426::GAL1-ILV5+GPM-kivD(described in Example 17) were transformed into Saccharomyces cerevisiaeYJR148w to produce strainYJR148w/pRS425::CUP1-alsS+FBA-ILV3/pRS426::GAL1-ILV5+GPM-kivD. A controlstrain was prepared by transforming vectors pRS425 and pRS426 (describedin Example 17) into Saccharomyces cerevisiae YJR148w (strainYJR148w/pRS425/pRS426). Strains were maintained on synthetic completemedium, as described in Example 18.

For isobutanol production, cells were transferred to synthetic completemedium containing 2% galactose and 1% raffinose, and lacking uracil andleucine. Aerobic and low oxygen cultures were prepared as described inExample 18. Approximately 12 h after inoculation, the inducer CuSO₄ wasadded up to a final concentration of 0.5 mM. Control cultures for eachstrain without CuSO₄ addition were also prepared. Culture supernatantswere sampled 23 h after CuSO₄ addition for determination of isobutanolby HPLC, as described in Example 18. The results are presented in Table11. Due to the widely different final optical densities observed andassociated with quantifying the residual carbon source, theconcentration of isobutanol per OD₆₀₀ unit (instead of molarselectivities) is provided in the table to allow comparison of strainscontaining the isobutanol biosynthetic pathway genes with the controls.

TABLE 11 Production of Isobutanol by S. cerevisiae YJR148w/pRS425::CUP1-alsS + FBA-ILV3/pRS426::GAL1-ILV5 + GPM-kivD Grown on Galactose andRaffinose CuSO₄, Isobutanol mM Isobutanol Strain O₂ level mM mM per ODunit YJR148w/pRS425/pRS426 (control) Aerobic 0.1 0.12 0.01YJR148w/pRS425/pRS426 (control) Aerobic 0.5 0.13 0.01YJR148w/pRS425::CUP1-alsS + Aerobic 0 0.20 0.03FBA-ILV3/pRS426::GAL1-ILV5 + GPM-kivD a YJR148w/pRS425::CUP1-alsS +Aerobic 0.03 0.82 0.09 FBA-ILV3/pRS426::GAL1-ILV5 + GPM-kivD bYJR148w/pRS425::CUP1-alsS + Aerobic 0.1 0.81 0.09FBA-ILV3/pRS426::GAL1-ILV5 + GPM-kivD c YJR148w/pRS425::CUP1-alsS +Aerobic 0.5 0.16 0.04 FBA-ILV3/pRS426::GAL1-ILV5 + GPM-kivD dYJR148w/pRS425::CUP1-alsS + Aerobic 0.5 0.18 0.01FBA-ILV3/pRS426::GAL1-ILV5 + GPM-kivD e YJR148w/pRS425/pRS426 (control)Low 0.1 0.042 0.007 YJR148w/pRS425/pRS426 (control) Low 0.5 0.023 0.006YJR148w/pRS425::CUP1-alsS + Low 0 0.1 0.04 FBA-ILV3/pRS426::GAL1-ILV5 +GPM-kivD a YJR148w/pRS425::CUP1-alsS + Low 0.03 0.024 0.02FBA-ILV3/pRS426::GAL1-ILV5 + GPM-kivD b YJR148w/pRS425::CUP1-alsS + Low0.1 0.030 0.04 FBA-ILV3/pRS426::GAL1-ILV5 + GPM-kivD cYJR148w/pRS425::CUP1-alsS + Low 0.5 0.008 0.02FBA-ILV3/pRS426::GAL1-ILV5 + GPM-kivD d YJR148w/pRS425::CUP1-alsS + Low0.5 0.008 0.004 FBA-ILV3/pRS426::GAL1-ILV5 + GPM-kivD e Strain suffixes“a”, “b”, “c”, “d” and “e” indicate separate isolates.

The results indicate that in general, higher levels of isobutanol peroptical density unit were produced by theYJR148w/pRS425::CUP1-alsS+FBA-ILV3/pRS426::GAL1-ILV5+GPM-kivD straincompared to the control strain under both aerobic and low oxygenconditions.

Example 20 Expression of an Isobutanol Biosynthetic Pathway in Bacillussubtilis

The purpose of this Example was to express an isobutanol biosyntheticpathway in Bacillus subtilis. The five genes of the isobutanol pathway(pathway steps (a) through (e) in FIG. 1) were split into two operonsfor expression. The three genes budB, ilvD, and kivD, encodingacetolactate synthase, acetohydroxy acid dehydratase, and branched-chainketo acid decarboxylase, respectively, were integrated into thechromosome of B. subtilis BE1010 (Payne and Jackson, J. Bacteriol.173:2278-2282 (1991)). The two genes ilvC and bdhB, encodingacetohydroxy acid isomeroreductase and butanol dehydrogenase,respectively, were cloned into an expression vector and transformed intothe Bacillus strain carrying the integrated isobutanol genes.

Integration of the Three Genes, budB, ilvD and kivD into the Chromosomeof B. subtilis BE1010.

Bacillus integration vectors pFP988DssPspac and pFP988DssPgroE were usedfor the chromosomal integration of the three genes, budB (SEQ ID NO:1),ilvD (SEQ ID NO:5), and kivD (SEQ ID NO:7). Both plasmids contain an E.coli replicon from pBR322, an ampicillin antibiotic marker for selectionin E. coli and two sections of homology to the sacB gene in the Bacilluschromosome that direct integration of the vector and interveningsequence by homologous recombination. Between the sacB homology regionsis a spac promoter (PgroE) on pFP988DssPspac or a groEL promoter (PgroE)on pFP988DssPgroE, and a selectable marker for Bacillus, erythromycin.The promoter region also contains the lacO sequence for regulation ofexpression by a lacI repressor protein. The sequences of pFP988DssPspac(6,341 bp) and pFP988DssPgroE (6,221 bp) are given as SEQ ID NO:142 andSEQ ID NO:143 respectively.

The cassette with three genes budB-ilvD-kivD was constructed by deletingthe ilvC gene from plasmid pTrc99a budB-ilvC-ilvD-kivD. The constructionof the plasmid pTrc99A::budB-ilvC-ilvD-kivD is described in Example 14.Plasmid pTrc99A::budB-ilvC-ilvD-kivD was digested with AflII and NheI,treated with the Klenow fragment of DNA polymerase to make blunt ends,and the resulting 9.4 kbp fragment containing pTrc99a vector, budB,ilvD, and kivD was gel-purified. The 9.4 kbp vector fragment wasself-ligated to create pTrc99A::budB-ilvD-kivD, and transformed intoDH5α competent cells (Invitrogen). A clone of pTrc99a budB-ilvD-kivD wasconfirmed for the ilvC gene deletion by restriction mapping. Theresulting plasmid pTrc99A::budB-ilvD-kivD was digested with SacI andtreated with the Klenow fragment of DNA polymerase to make blunt ends.The plasmid was then digested with BamHI and the resulting 5,297 bpbudB-ilvD-kivD fragment was gel-purified. The 5,297 bp budB-ilvD-kivDfragment was ligated into the SmaI and BamHI sites of the integrationvector pFP988DssPspac. The ligation mixture was transformed into DH5αcompetent cells. Transformants were screened by PCR amplification of the5.3 kbp budB-ilvD-kivD fragment with primers T-budB(BamHI) (SEQ IDNO:144) and B-kivD(BamHI) (SEQ ID NO:145). The correct clone was namedpFP988DssPspac-budB-ilvD-kivD.

Plasmid pFP988DssPspac-budB-ilvD-kivD was prepared from the E. colitransformant, and transformed into B. subtilis BE1010 competent cells,which had been prepared as described by Doyle et al. (J. Bacteriol.144:957 (1980)). Competent cells were harvested by centrifugation andthe cell pellets were resuspended in a small volume of the supernatant.To one volume of competent cells, two volumes of SPII-EGTA medium(Methods for General and Molecular Bacteriology, P. Gerhardt et al.,Ed., American Society for Microbiology, Washington, D.C. (1994)) wasadded. Aliquots (0.3 mL) of cells were dispensed into test tubes andthen 2 to 3 μg of plasmid pFP988DssPspac-budB-ilvD-kivD was added to thetubes. The tubes were incubated for 30 min at 37° C. with shaking, afterwhich 0.1 mL of 10% yeast extract was added to each tube and they werefurther incubated for 60 min. Transformants were grown for selection onLB plates containing erythromycin (1.0 μg/mL) using the double agaroverlay method (Methods for General and Molecular Bacteriology, supra).Transformants were screened by PCR amplification with primers N130SeqF1(SEQ ID NO:40) and N130SeqR1 (SEQ ID NO:44) for budB, and N133SeqF1 (SEQID NO:62) and N133SeqR1 (SEQ ID NO:66) for kivD. Positive integrantsshowed the expected 1.7 kbp budB and 1.7 kbp kivD PCR products. Twopositive integrants were identified and named B. subtilis BE1010ΔsacB::Pspac-budB-ilvD-kivD #2-3-2 and B. subtilis BE1010ΔsacB::Pspac-budB-ilvD-kivD #6-12-7.

Assay of the enzyme activities in integrants B. subtilis BE1010ΔsacB::Pspac-budB-ilvD-kivD #2-3-2 and B. subtilis BE1010ΔsacB::Pspac-budB-ilvD-kivD #6-12-7 indicated that the activities ofBudB, IlvD and KivD were low under the control of the spac promoter(Pspac). To improve expression of functional enzymes, the Pspac promoterwas replaced by a PgroE promoter from plasmid pHT01 (MoBitec,Goettingen, Germany).

A 6,039 bp pFP988Dss vector fragment, given as SEQ ID NO:146, wasexcised from an unrelated plasmid by restriction digestion with XhoI andBamHI, and was gel-purified. The PgroE promoter was PCR-amplified fromplasmid pHT01 with primers T-groE(XhoI) (SEQ ID NO:147) andB-groEL(SpeI,BamH1) (SEQ ID NO:148). The PCR product was digested withXhoI and BamHI, ligated with the 6,039 bp pFP988Dss vector fragment, andtransformed into DH5α competent cells. Transformants were screened byPCR amplification with primers T-groE(XhoI) and B-groEL(SpeI,BamH1).Positive clones showed the expected 174 bp PgroE PCR product and werenamed pFP988DssPgroE. The plasmid pFP988DssPgroE was also confirmed byDNA sequence.

Plasmid pFP988DssPspac-budB-ilvD-kivD was digested with SpeI and Pmeland the resulting 5,313 bp budB-ilvD-kivD fragment was gel-purified. ThebudB-ilvD-kivD fragment was ligated into SpeI and Pmel sites ofpFP988DssPgroE and transformed into DH5α competent cells. Positiveclones were screened for a 1,690 bp PCR product by PCR amplificationwith primers T-groEL (SEQ ID NO:149) and N111 (SEQ ID NO:20). Thepositive clone was named pFP988DssPgroE-budB-ilvD-kivD.

Plasmid pFP988DssPgroE-budB-ilvD-kivD was prepared from the E. colitransformant, and transformed into Bacillus subtilis BE1010 competentcells as described above. Transformants were screened by PCRamplification with primers N130SeqF1 (SEQ ID NO:40) and N130SeqR1 (SEQID NO:44) for budB, and N133SegF1 (SEQ ID NO:62) and N133SeqR1 (SEQ IDNO:66) for kivD. Positive integrants showed the expected 1.7 kbp budBand 1.7 kbp kivD PCR products. Two positive integrants were isolated andnamed B. subtilis BE1010 ΔsacB::PgroE-budB-ilvD-kivD #1-7 and B.subtilis BE1010 ΔsacB::PgroE-budB-ilvD-kivD #8-16.

Plasmid Expression of ilvC and bdhB Genes.

Two remaining isobutanol genes, ilvC and bdhB, were expressed from aplasmid. Plasmid pHT01 (MoBitec), a Bacillus-E. coli shuttle vector, wasused to fuse an ilvC gene from B. subtilis to a PgroE promoter so thatthe ilvC gene was expressed from the PgroE promoter containing a lacOsequence. The ilvC gene, given as SEQ ID NO:186, was PCR-amplified fromB. subtilis BR151 (ATCC 33677) genomic DNA with primersT-ilvCB.s.(BamHI) (SEQ ID NO:150) and B-ilvCB.s.(SpeI BamHI) (SEQ IDNO:151). The 1,067 bp ilvC PCR product was digested with BamHI andligated into the BamHI site of pHT01. The ligation mixture wastransformed into DH5α competent cells. Positive clones were screened fora 1,188 bp PCR product by PCR amplification with primers T-groEL andB-ilvB.s.(SpeI BamHI). The positive clone was named pHT01-ilvC(B.s).Plasmid pHT01-ilvC(B.s) was used as a template for PCR amplification ofthe PgroE-ilvC fused fragment.

Plasmid pBD64 (Minton et al., Nucleic Acids Res. 18:1651 (1990)) is afairly stable vector for expression of foreign genes in B. subtilis andcontains a repB gene and chloramphenicol and kanamycin resistance genesfor selection in B. subtilis. This plasmid was used for expression ofilvC and bdhB under the control of a PgroE promoter. To clonePgroE-ilvC, bdhB and a lacI repressor gene into plasmid pBD64, aone-step assembly method was used (Tsuge et al., Nucleic Acids Res.31:e133 (2003)). A 3,588 bp pBD64 fragment containing a repB gene, whichincluded the replication function, and the kanamycin antibiotic markerwas PCR-amplified from pBD64 with primers T-BD64(DraIII) (SEQ IDNO:152), which introduced a DraIII sequence (CACCGAGTG), andB-BD64(DraIII) (SEQ ID NO:153), which introduced a DraIII sequence(CACCTGGTG). A 1,327 bp lacI repressor gene was PCR-amplified frompMUTIN4 (Vagner et al., Microbiol. 144:3097-3104 (1998)) withT-lacIq(DraIII) (SEQ ID NO:154), which introduced a DraIII sequence(CACCAGGTG) and B-lacIq(DraIII) (SEQ ID NO:155), which introduced aDraIII sequence (CACGGGGTG). A 1,224 bp PgroE-ilvC fused cassette wasPCR-amplified from pHT01-ilvC(B.s) with T-groE(DraIII) (SEQ ID NO:156),which introduced a DraIII sequence (CACCCCGTG), and B-B.s.ilvC(DraIII)(SEQ ID NO:157), which introduced a DraIII sequence (CACCGTGTG). A 1.2kbp bdhB gene (SEQ ID NO:158) was PCR-amplified from Clostridiumacetobutylicum (ATCC 824) genomic DNA with primers T-bdhB(DraIII) (SEQID NO:159), which introduced a DraIII sequence (CACACGGTG), andB-bdhB(rrnBT1DraIII) (SEQ ID NO:160), which introduced a DraIII sequence(CACTCGGTG). The three underlined letters in the variable region of theDraIII recognition sequences were designed for specific base-pairing toassemble the four fragments with an order of pBD64-lacI-PgroEilvC-bdhB.Each PCR product with DraIII sites at both ends was digested separatelywith DraIII, and the resulting DraIII fragments, 3,588 bp pBD64, lacI,PgroEilvC, and bdhB were gel-purified using a QIAGEN gel extraction kit(QIAGEN). A mixture containing an equimolar concentration of eachfragment with a total DNA concentration of 30 to 50 μg/100 μL wasprepared for ligation. The ligation solution was then incubated at 16°C. overnight. The ligation generated high molecular weight tandem repeatDNA. The ligated long, linear DNA mixture was directly transformed intocompetent B. subtilis BE1010, prepared as described above. B. subtilispreferentially takes up long repeated linear DNA forms, rather thancircular DNA to establish a plasmid. After transformation the culturewas spread onto an LB plate containing 10 μg/mL of kanamycin forselection. Positive recombinant plasmids were screened by DraIIIdigestion, giving four fragments with an expected size of 3,588 bp(pBD64), 1,327 bp (lacI), 1,224 bp (PgorE-ilvC), and 1,194 bp (bdhB).The positive plasmid was named pBDPgroE-ilvC(B.s.)-bdhB.

Demonstration of Isobutanol Production from Glucose or Sucrose by B.subtilis BE1010 ΔsacB::PqroE-budB-ilvD-kivD/pBDPqroE-ilvC(B.s.)-bdhB.

To construct the recombinant B. subtilis expressing the five genes ofthe isobutanol biosynthetic pathway, competent cells of the twointegrants B. subtilis BE1010 ΔsacB-PgroE-budB-ilvD-kivD #1-7 and B.subtilis BE1010 ΔsacB::PgroE-budB-ilvD-kivD #8-16 were prepared asdescribed above, and transformed with plasmid pBDPgroE-ilvC(B.s.)-bdhB,yielding B. subtilis BE1010 ΔsacB::PgroE-budB-ilvD-kivD#1-7/pBDPgroE-ilvC(B.s.)-bdhB and B. subtilis BE1010ΔsacB::PgroE-budB-ilvD-kivD #8-16/pBDPgroE-ilvC(B.s.)-bdhB.

The two recombinant strains were inoculated in either 25 mL or 100 mL ofglucose medium containing kanamycin (10 μg/mL) in 125 mL flasks tosimulate high and low oxygen conditions, respectively, and aerobicallygrown at 37° C. with shaking at 200 rpm. The medium consisted of 10 mM(NH₄)₂SO₄, 5 mM potassium phosphate buffer (pH 7.0), 100 mM MOPS/KOHbuffer (pH 7.0), 20 mM glutamic acid/KOH (pH 7.0), 2% S10 metal mix, 1%glucose, 0.01% yeast extract, 0.01% casamino acids, and 50 μg/mL each ofL-tryptophan, L-methionine, and L-lysine. The S10 metal mix consisted of200 mM MgCl₂, 70 mM CaCl₂, 5 mM MnCl₂, 0.1 mM FeCl₃, 0.1 mM ZnCl₂, 0.2mM thiamine hydrochloride, 0.172 mM CuSO₄, 0.253 mM CoCl₂, and 0.242 mMNa₂MoO₄. The cells were induced with 1.0 mMisopropyl-β-D-thiogalactopyranoiside (IPTG) at early-log phase (OD₆₀₀ ofapproximately 0.2). At 24 h after inoculation, an aliquot of the brothwas analyzed by HPLC (Shodex Sugar SH1011 column) with refractive index(RI) detection for isobutanol content, as described in the GeneralMethods section. The HPLC results are shown in Table 12.

TABLE 12 Production of Isobutanol from Glucose by B. subtilis BE1010ΔsacB::PgroE-budB-ilvD-kivD/pBDPgroE-ilvC(B.s.)-bdhB Strains isobutanol,molar selectivity, Strain O₂ Level mM % B. subtilis a high 1.00 1.8(induced) B. subtilis b high 0.87 1.6 (induced) B. subtilis a low 0.060.1 (induced) B. subtilis b low 0.14 0.3 (induced) B. subtilis a is B.subtilis BE1010 ΔsacB::PgroE-budB-ilvD-kivD#1-7/pBDPgroE-ilvC(B.s.)-bdhB B. subtilis b is B. subtilis BE1010ΔsacB::PgroE-budB-ilvD-kivD #8-16/pBDPgroE-ilvC(B.s.)-bdhB

The isolate of B. subtilis BE1010 ΔsacB::PgroE-budB-ilvD-kivD#1-7/pBDPgroE-ilvC(B.s.)-bdhB was also examined for isobutanolproduction from sucrose, essentially as described above. The recombinantstrain was inoculated in 25 mL or 75 mL of sucrose medium containingkanamycin (10 μg/mL) in 125 mL flasks to simulate high and medium oxygenlevels, and grown at 37° C. with shaking at 200 rpm. The sucrose mediumwas identical to the glucose medium except that glucose (10 g/L) wasreplaced with 10 g/L of sucrose. The cells were uninduced, or inducedwith 1.0 mM isopropyl-β-D-thiogalactopyranoiside (IPTG) at early-logphase (OD₆₀₀ of approximately 0.2). At 24 h after inoculation, analiquot of the broth was analyzed by HPLC (Shodex Sugar SH1011 column)with refractive index (RI) detection for isobutanol content, asdescribed in the General Methods section. The HPLC results are given inTable 13.

TABLE 13 Production of Isobutanol from Sucrose by B. subtilis StrainBE1010 ΔsacB::PgroE-budB-ilvD-kivD/pBDPgroE-ilvC(B.s.)-bdhB Strain O₂Level isobutanol, mM molar selectivity, % B. subtilis a high Notdetected Not detected (uninduced) B. subtilis a high 0.44 4.9 (induced)B. subtilis a medium 0.83 8.6 (induced) B. subtilis a is B. subtilisBE1010 ΔsacB::PgroE-budB-ilvD-kivD #1-7/pBDPgroE-ilvC(B.s.)-bdhB

Example 21 (Prophetic) Expression of an Isobutanol Biosynthetic Pathwayin Lactobacillus plantarum

The purpose of this prophetic Example is to describe how to express anisobutanol biosynthetic pathway in Lactobacillus plantarum. The fivegenes of the isobutanol pathway, encoding five enzyme activities, aredivided into two operons for expression. The budB, ilvD and kivD genes,encoding the enzymes acetolactate synthase, acetohydroxy aciddehydratase, and branched-chain α-keto acid decarboxylase, respectively,are integrated into the chromosome of Lactobacillus plantarum byhomologous recombination using the method described by Hols et al.(Appl. Environ. Microbiol. 60:1401-1413 (1994)). The remaining two genes(ilvC and bdhB, encoding the enzymes acetohydroxy acid reductoisomeraseand butanol dehydrogenase, respectively) are cloned into an expressionplasmid and transformed into the Lactobacillus strain carrying theintegrated isobutanol genes. Lactobacillus plantarum is grown in MRSmedium (Difco Laboratories, Detroit, Mich.) at 37° C., and chromosomalDNA is isolated as described by Moreira et al. (BMC Microbiol. 5:15(2005)).

Integration.

The budB-ilvD-kivD cassette under the control of the synthetic P11promoter (Rud et al., Microbiology 152:1011-1019 (2006)) is integratedinto the chromosome of Lactobacillus plantarum ATCC BAA-793 (NCIMB 8826)at the IdhL1 locus by homologous recombination. To build the ldhLintegration targeting vector, a DNA fragment from Lactobacillusplantarum (Genbank NC_(—)004567) with homology to ldhL is PCR amplifiedwith primers LDH EcoRV F (SEQ ID NO:161) and LDH AatIIR (SEQ ID NO:162).The 1986 bp PCR fragment is cloned into pCR4Blunt-TOPO and sequenced.The pCR4Blunt-TOPO-IdhL1 clone is digested with EcoRV and AatIIreleasing a 1982 bp IdhL1 fragment that is gel-purified. The integrationvector pFP988, given as SEQ ID NO:177, is digested with HindIII andtreated with Klenow DNA polymerase to blunt the ends. The linearizedplasmid is then digested with AatII and the 2931 bp vector fragment isgel purified. The EcoRV/AatII IdhL1 fragment is ligated with the pFP988vector fragment and transformed into E. coli Top10 cells. Transformantsare selected on LB agar plates containing ampicillin (100 μg/mL) and arescreened by colony PCR to confirm construction of pFP988-IdhL.

To add a selectable marker to the integrating DNA, the Cm gene with itspromoter is PCR amplified from pC194 (GenBank NC_(—)002013, SEQ IDNO:267) with primers Cm F (SEQ ID NO:163) and Cm R (SEQ ID NO:164),amplifying a 836 bp PCR product. This PCR product is cloned intopCR4Blunt-TOPO and transformed into E. coli Top10 cells, creatingpCR4Blunt-TOPO-Cm. After sequencing to confirm that no errors areintroduced by PCR, the Cm cassette is digested from pCR4Blunt-TOPO-Cm asan 828 bp MluI/SwaI fragment and is gel purified. The IdhL-homologycontaining integration vector pFP988-IdhL is digested with MluI and SwaIand the 4740 bp vector fragment is gel purified. The Cm cassettefragment is ligated with the pFP988-IdhL vector creatingpFP988-DldhL::Cm.

Finally the budB-ilvD-kivD cassette from pFP988DssPspac-budB-ilvD-kivD,described in Example 20, is modified to replace the amylase promoterwith the synthetic P11 promoter. Then, the whole operon is moved intopFP988-DldhL::Cm. The P11 promoter is built by oligonucleotide annealingwith primer P11 F-Stul (SEQ ID NO:165) and P11 R-SpeI (SEQ ID NO:166).The annealed oligonucleotide is gel-purified on a 6% Ultra PAGE gel(Embi Tec, San Diego, Calif.). The plasmidpFP988DssPspac-budB-ilvD-kivD, containing the amylase promoter, isdigested with Stul and SpeI and the resulting 10.9 kbp vector fragmentis gel-purified. The isolated P11 fragment is ligated with the digestedpFP988DssPspac-budB-ilvD-kivD to create pFP988-P11-budB-ilvD-kivD.Plasmid pFP988-P11-budB-ilvD-kivD is then digested with Stul and BamHIand the resulting 5.4 kbp P11-budB-ilvD-kivD fragment is gel-purified.pFP988-DldhL::Cm is digested with Hpal and BamHI and the 5.5 kbp vectorfragment isolated. The budB-ilvD-kivD operon is ligated with theintegration vector pFP988-DldhL::Cm to createpFP988-DldhL-P11-budB-ilvD-kivD::Cm.

Integration of pFP988-DldhL-P11-budB-ilvD-kivD::Cm into L. plantarumBAA-793 to Form L. plantarum ΔldhL1::budB-ilvD-kivD::Cm ComprisingExogenous budB, ilvD, and kivD Genes.

Electrocompetent cells of L. plantarum are prepared as described byAukrust, T. W., et al. (In: Electroporation Protocols forMicroorganisms; Nickoloff, J. A., Ed.; Methods in Molecular Biology,Vol. 47; Humana Press, Inc., Totowa, N.J., 1995, pp 201-208). Afterelectroporation, cells are outgrown in MRSSM medium (MRS mediumsupplemented with 0.5 M sucrose and 0.1 M MgCl₂) as described by Aukrustet al. supra for 2 h at 37° C. without shaking. Electroporated cells areplated for selection on MRS plates containing chloramphenicol (10 μg/mL)and incubated at 37° C. Transformants are initially screened by colonyPCR amplification to confirm integration, and initial positive clonesare then more rigorously screened by PCR amplification with a battery ofprimers.

Plasmid Expression of ilvC and bdhB Genes.

The remaining two isobutanol genes are expressed from plasmid pTRKH3(O'Sullivan D J and Klaenhammer TR, Gene 137:227-231 (1993)) under thecontrol of the L. plantarum IdhL promoter (Ferain et al., J. Bacteriol.176:596-601 (1994)). The IdhL promoter is PCR amplified from the genomeof L. plantarum ATCC BAA-793 using primers PldhL F-HindIII (SEQ IDNO:167) and PldhL R-BamHI (SEQ ID NO:168). The 411 bp PCR product iscloned into pCR4Blunt-TOPO and sequenced. The resulting plasmid,pCR4Blunt-TOPO-PldhL is digested with HindIII and BamHI releasing thePldhL fragment.

Plasmid pTRKH3 is digested with HindIII and SphI and the gel-purifiedvector fragment is ligated with the PldhL fragment and the gel-purified2.4 kbp BamHI/SphI fragment containing ilvC(B.s.)-bdhB from the Bacillusexpression plasmid pBDPgroE-ilvC(B.s.)-bdhB (Example 20) in a three-wayligation. The ligation mixture is transformed into E. coli Top 10 cellsand transformants are grown on Brain Heart Infusion (BHI, DifcoLaboratories, Detroit, Mich.) plates containing erythromycin (150 mg/L).Transformants are screened by PCR to confirm construction. The resultingexpression plasmid, pTRKH3-ilvC(B.s.)-bdhB is transformed into L.plantarum ΔldhL1::budB-ilvD-kivD::Cm by electroporation, as describedabove.

L. plantarum ΔldhL1::budB-ilvD-kivD::Cm containingpTRKH3-ilvC(B.s.)-bdhB is inoculated into a 250 mL shake flaskcontaining 50 mL of MRS medium plus erythromycin (10 μg/mL) and grown at37° C. for 18 to 24 h without shaking, after which isobutanol isdetected by HPLC or GC analysis, as described in the General Methodssection.

Example 22 (Prophetic) Expression of an Isobutanol Biosynthetic Pathwayin Enterococcus faecalis

The purpose of this prophetic Example is to describe how to express anisobutanol biosynthetic pathway in Enterococcus faecalis. The completegenome sequence of Enterococcus faecalis strain V583, which is used asthe host strain for the expression of the isobutanol biosyntheticpathway in this Example, has been published (Paulsen et al., Science299:2071-2074 (2003)). An E. coli/Gram-positive shuttle vector, PlasmidpTRKH3 (O'Sullivan D J and Klaenhammer T R, Gene 137:227-231 (1993)), isused for expression of the five genes (budB, ilvC, ilvD, kivD, bdhB) ofthe isobutanol pathway in one operon. pTRKH3 contains an E. coli plasmidp15A replication origin, the pAMβ1 replicon, and two antibioticresistance selection markers for tetracycline and erythromycin.Tetracycline resistance is only expressed in E. coli, and erythromycinresistance is expressed in both E. coli and Gram-positive bacteria.Plasmid pAMβ1 derivatives can replicate in E. faecalis (Poyart et al.,FEMS Microbiol. Lett. 156:193-198 (1997)). The inducible nisA promoter(PnisA), which has been used for efficient control of gene expression bynisin in a variety of Gram-positive bacteria including Enterococcusfaecalis (Eichenbaum et al., Appl. Environ. Microbiol. 64:2763-2769(1998)), is used to control expression of the five desired genesencoding the enzymes of the isobutanol biosynthetic pathway.

The plasmid pTrc99A::budB-ilvC-ilvD-kivD (described in Example 14),which contains the isobutanol pathway operon, is modified to replace theE. coli ilvC gene (SEQ ID NO:3) with the B. subtilis ilvC gene (SEQ IDNO:184). Additionally, the bdhB gene (SEQ ID NO:158) from Clostridiumacetobutylicum is added to the end of the operon. First, the bdhB genefrom pBDPgroE-ilvC(B.s.)-bdhB (described in Example 20) is amplifiedusing primers F-bdhB-AvrII (SEQ ID NO:169) and R-bdhB-BamHI (SEQ IDNO:170), and then TOPO cloned and sequenced. The 1194 bp bdhB fragmentis isolated by digestion with AvrII and BamHI, followed by gelpurification. This bdhB fragment is ligated withpTrc99A::budB-ilvC-ilvD-kivD that has previously been digested withAvrII and BamHI and the resulting fragment is gel purified. The ligationmixture is transformed into E. coli Top10 cells by electroporation andtransformants are selected following overnight growth at 37° C. on LBagar plates containing ampicillin (100 μg/mL). The transformants arethen screened by colony PCR to confirm the correct clone containingpTrc99A::budB-ilvC-ilvD-kivD-bdhB.

Next, ilvC(B.s.) is amplified from pBDPgroE-ilvC(B.s.)-bdhB (describedin Example 20) using primers F-ilvC(B.s.)-AflII (SEQ ID NO:171) andR-ilvC(B.s.)-NotI (SEQ ID NO:172). The PCR product is TOPO cloned andsequenced. The 1051 bp ilvC(B.s.) fragment is isolated by digestion withAflII and NotI followed by gel purification. This fragment is ligatedwith pTrc99A::budB-ilvC-ilvD-kivD-bdhB that has been cut with AflII andNotI to release the E. coli ilvC (the 10.7 kbp vector band is gelpurified prior to ligation with ilvC(B.s.)). The ligation mixture istransformed into E. coli Top10 cells by electroporation andtransformants are selected following overnight growth at 37° C. on LBagar plates containing ampicillin (100 μg/mL). The transformants arethen screened by colony PCR to confirm the correct clone containingpTrc99A::budB-ilvC(B.s.)-ilvD-kivD-bdhB.

To provide a promoter for the E. coli/Gram-positive shuttle vectorpTRKH3, the nisA promoter (Chandrapati et al., Mol. Microbiol.46(2):467-477 (2002)) is PCR-amplified from Lactococcus lactis genomicDNA with primers F-PnisA(HindIII) (SEQ ID NO:173) and R-PnisA(SpeIBamHI) (SEQ ID NO:174) and then TOPO cloned. After sequencing, the 213bp nisA promoter fragment is isolated by digestion with HindIII andBamHI followed by gel purification. Plasmid pTRKH3 is digested withHindIII and BamHI and the vector fragment is gel-purified. Thelinearized pTRKH3 is ligated with the PnisA fragment and transformedinto E. coli Top10 cells by electroporation. Transformants are selectedfollowing overnight growth at 37° C. on LB agar plates containingerythromycin (25 μg/mL). The transformants are then screened by colonyPCR to confirm the correct clone of pTRKH3-PnisA.

Plasmid pTRKH3-PnisA is digested with SpeI and BamHI, and the vector isgel-purified. Plasmid pTrc99A::budB-ilvC(B.s)-ilvD-kivD-bdhB, describedabove, is digested with SpeI and BamHI, and the 7.5 kbp fragment isgel-purified. The 7.5 kbp budB-ilvC(B.s)-ilvD-kivD-bdhB fragment isligated into the pTRKH3-PnisA vector at the SpeI and BamHI sites. Theligation mixture is transformed into E. coli Top10 cells byelectroporation and transformants are selected following overnightgrowth on LB agar plates containing erythromycin (25 μg/mL) at 37° C.The transformants are then screened by colony PCR. The resulting plasmidis named pTRKH3-PnisA-budB-ilvC(B.s)-ilvD-kivD-bdhB. This plasmid isprepared from the E. coli transformants and transformed intoelectrocompetent E. faecalis V583 cells by electroporation using methodsknown in the art (Aukrust, T. W., et al. In: Electroporation Protocolsfor Microorganisms; Nickoloff, J. A., Ed.; Methods in Molecular Biology,Vol. 47; Humana Press, Inc., Totowa, N.J., 1995, pp 217-226), resultingin E. faecalis V583/pTRKH3-PnisA-budB-ilvC(B.s)-ilvD-kivD-bdhB.

The second plasmid containing nisA regulatory genes, nisR and nisK, theadd9 spectinomycin resistance gene, and the pSH71 origin of replicationis transformed into E. faecalisV583/pTRKH3-PnisA-budB-ilvC(B.s)-ilvD-kivD-bdhB by electroporation. Theplasmid containing pSH71 origin of replication is compatible with pAMJ31derivatives in E. faecalis (Eichenbaum et al., supra). Double drugresistant transformants are selected on LB agar plates containingerythromycin (25 μg/mL) and spectinomycin (100 μg/mL), grown at 37° C.

The resulting E. faecalis strain V5838 harboring two plasmids, i.e., anexpression plasmid (pTRKH3-PnisA-budB-ilvC(B.s)-ilvD-kivD-bdhB) and aregulatory plasmid (pSH71-nisRK), is inoculated into a 250 mL shakeflask containing 50 mL of Todd-Hewitt broth supplemented with yeastextract (0.2%) (Fischetti et al., J. Exp. Med. 161:1384-1401 (1985)),nisin (20 μg/mL) (Eichenbaum et al., supra), erythromycin (25 μg/mL),and spectinomycin (100 μg/mL). The flask is incubated without shaking at37° C. for 18-24 h, after which time, isobutanol production is measuredby HPLC or GC analysis, as described in the General Methods section.

What is claimed is:
 1. A method comprising: (a) determining theacetohydroxy acid isomeroreductase (KARI) activity of a cell freeextract prepared from a host cell expressing a heterologous KARI enzyme;and (b) expressing a heterologous DNA molecule encoding the KARI enzymein a recombinant microorganism expressing an engineered isobutanolbiosynthetic pathway, wherein the cell free extract comprises higherKARI activity than a control cell free extract prepared from a host cellnot expressing the heterologous KARI enzyme, and wherein the recombinantmicroorganism is capable of bioproducing isobutanol suitable for use infuel cells or vehicles.
 2. The method of claim 1, wherein the host cellof (a) is E. coli.
 3. The method of claim 1, wherein the recombinantmicroorganism is selected from the group consisting of: a bacterium, acyanobacterium, a filamentous fungus and a yeast.
 4. The method of claim3, wherein the recombinant microorganism is yeast.
 5. The method ofclaim 1, wherein the recombinant microorganism is Saccharomycescerevisiae.
 6. The method of claim 1, wherein the recombinantmicroorganism using glucose as a carbon source produces more isobutanolthan a control microorganism not expressing the engineered biosyntheticisobutanol pathway.
 7. The method of claim 1, wherein the recombinantmicroorganism comprises an inactivated gene that reduces yield loss fromcompeting pathways for carbon flow.
 8. The method of claim 1, whereinthe engineered biosynthetic isobutanol pathway comprises an acetolactatesynthase (ALS) enzyme, a KARI enzyme, an acetohydroxy acid dehydratase(DHAD) enzyme, a branched chain keto acid decarboxylase enzyme, and abranched-chain alcohol dehydrogenase (ADH) enzyme, each of which isencoded by a heterologous DNA molecule.
 9. The method of claim 1,wherein the engineered biosynthetic isobutanol pathway comprises an ALSenzyme, a KARI enzyme, a DHAD enzyme, a branched-chain keto aciddehydrogenase enzyme, an acylating aldehyde dehydrogenase enzyme, and anADH enzyme, each of which is encoded by a heterologous DNA molecule. 10.The method of claim 1, wherein the engineered biosynthetic isobutanolpathway comprises an ALS enzyme, a KARI enzyme, a DHAD enzyme, at leastone of a transaminase enzyme and a valine dehydrogenase enzyme, a valinedecarboxylase enzyme, an omega transaminase enzyme, and an ADH enzyme,each of which is encoded by a heterologous DNA molecule.
 11. The methodof claim 8, wherein the branched chain keto acid decarboxylase enzymehas 50-200-fold preference for ketoisovalerate over pyruvate.
 12. Themethod of claim 8, wherein the ALS, KARI, DHAD, branched chain keto aciddecarboxylase, and ADH enzymes each lack a mitochondrial targetingsequence.
 13. The method of claim 8, wherein the ALS enzyme has an ECnumber of 2.2.1.6, the KARI enzyme has an EC number of 1.1.1.86, theDHAD enzyme has an EC number of 4.2.1.9, the branched chain keto aciddecarboxylase enzyme has an EC number of 4.1.1.72, and the ADH enzymehas an EC number of 1.1.1.1, 1.1.1.2, or 1.1.1.265.
 14. The method ofclaim 13, wherein the ALS enzyme has an amino acid sequence selectedfrom SEQ ID NOs: 2, 178, and
 180. 15. The method of claim 13, whereinthe KARI enzyme has an amino acid sequence selected from SEQ ID NOs: 4,181, 183, and
 185. 16. The method of claim 13, wherein the DHAD enzymehas an amino acid sequence selected from SEQ ID NOs: 6, 186, 188, and190.
 17. The method of claim 13, wherein the branched chain keto aciddecarboxylase enzyme has an amino acid sequence selected from SEQ IDNOs: 8, 193, 195, and
 197. 18. The method of claim 1, wherein therecombinant microorganism is capable of bioproducing isobutanol as asingle product.